Differential Impact of Vegf and Fgf2 Signaling Mechanisms on Flt1 Pre-Mrna Splicing

DIFFERENTIAL IMPACT OF VEGF AND FGF2 SIGNALING MECHANISMS ON FLT1 PRE-MRNA SPLICING

Laura Beth Payne

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Biomedical and Veterinary Sciences

William R. Huckle Carla Finkielstein Ian Herring William Eyestone

April 27, 2016

Blacksburg, Virginia

Keywords: Akt, Alternative splicing, FGF2, Flt1, ERK, Preeclampsia, pre-mRNA, Signal transduction, soluble Flt1, SR proteins, VEGF

DIFFERENTIAL IMPACT OF VEGF AND FGF2 SIGNALING MECHANISMS ON FLT1 PRE-MRNA SPLICING

Laura Beth Payne

ABSTRACT, Academic

The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC- independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These

findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation.

ABSTRACT, Public

About 95% of the genes in the human body are each processed in different ways to ultimately yield multiple different proteins – complex molecules that make up structural and functional features of cells and tissues of the body. In pathology and disease, cues from outside the cell may direct a particular gene to produce an incorrect protein or balance of proteins. One such case is preeclampsia - a pregnancy-related disorder affecting as many as 1 out of every 20 pregnancies in the US alone. Preeclampsia is characterized by hypertension and protein in the urine and can lead to organ failure and death of both mother and baby. Currently there is no treatment to effectively control the disorder during pregnancy, the only cure being delivery. The originating cause of preeclampsia remains unknown. What is known, however, is some of the cells in the placenta incorrectly express proteins from the gene called Flt1. Flt1 is protein that crosses through the outer layer of the cell (a cell-surface receptor) and binds vascular endothelial growth factor (VEGF), which controls growth of blood vessels. VEGF binds to two main receptors on the surface of the cell, which then relay the “signal” inside the cell, leading to the appropriate changes in cell function. The responses to VEGF signaling include blood vessel growth or branching. In the placenta, the VEGF signal, along with other signaling molecules such as fibroblast growth factor 2 (FGF2), directs blood vessels to grow properly in order to deliver nutrients and oxygen to the growing fetus. In preeclampsia, production (expression) of three protein receptors that come from the gene Flt1, called Flt1, sFlt1_v1 and sFlt1_v2, is imbalanced. In turn, the development of the blood vessels in the placenta is inadequate, leading to pregnancy complications. We investigated the influence of VEGF and FGF2 on the expression of the different forms (variants) of Flt1. We found that VEGF and FGF2 cause the different variants of Flt1 to be expressed differently. We investigated proteins inside the cells, by which they send (transduce) their signals, and found that VEGF and FGF2 use different but related proteins to influence Flt1 variant expression. We also examined SR proteins, which are directed by cues outside the cell (such as VEGF or FGF2) to directly interact with and influence which variants are expressed from a particular gene. We used prediction tools to identify specific SR proteins that may be directed by VEGF or FGF2 to bind to Flt1 before it is processed into different variants and, in turn, influence which variants are expressed. We identified two candidate SR proteins and found that FGF2 appears to affect the expression of one of them, SRSF3. Overall, VEGF and FGF2,

which are imperative for proper blood vessel growth in the placenta, differently affect the amounts of Ftl1 variants, likely by sending their signals through related but different proteins inside the cell. Additionally, our results indicate that FGF2 may influence Flt1 variant expression by activating SR proteins and, in the case of SRSF3, by affecting how much is expressed. Our findings provide new evidence for the role of FGF2 in preeclampsia and sheds light on how FGF2 and VEGF influence Flt1 variant expression. Ultimately, understanding how and why imbalanced Flt1 variant expression occurs in preeclampsia may lead to life-saving therapies where none currently exist. Our multi-level investigation of proteins that influence Flt1 variant expression, leading from outside the cell and into the nucleus, reveals different levels for further investigation and eventual targeted therapy pursuit.

DEDICATION

This manuscript is dedicated to my loving family for their unwavering support. To my parents, Bill and Bonnie Payne, thank you for sowing and nurturing the seeds of personal growth and optimism. You have made all my accomplishments possible. Thank you, from the bottom of my heart. I love you. To my sisters, thank you for believing in me and seeing my true heart. Such gifts have seen me through the days of doubt. You have my undying gratitude and love. To my parents-in-law, I am indebted to you for the incredibly gracious support you have given me during this journey. I offer my love and gratitude.

Finally, this manuscript is dedicated to my wonderful husband, John Eustis, and my magical daughter, Graelyn Eustis. To John, my thanks is unending – your support and encouragement have been monumental and foundational to my success. Thank you for being so solid and sharing your love with me. I love you very much. To Graelyn, words fall short – your very presence in my life has inspired me in so many ways. You truly can achieve whatever you set your sights on…but the most important achievement is remembering that life’s true meaning is found in the present, regardless of where you have been or where you are headed. My love for you is, and will always be, immeasurable and unconditional.

ACKNOWLEDGMENTS

Funding support was provided by an assistantship provided by the Virginia Maryland Regional College of Veterinary Medicine.

I thank my fellow BMVS members for allowing me to pick their brains and occasionally borrow the random reagent. I would also like to thank my fellow lab-mate, Erwin Kristobal Gudenschwager Basso, for allowing me to bump shoulders while we pursued our respective projects.

I thank my committee members for their support, encouragement, and expertise during the course of my graduate program. I am grateful for the generous time an effort you put forth for me.

Finally, I thank my advisor Dr. Huckle. Thank you so much for your calm and encouraging guidance as I learned the ropes throughout this process called graduate school. Thank you for allowing me to be an individual and a mom, while also wearing the hat of student. Most of all, thank you for believing in me and for sharing experiential words of direction… gifts that will live far beyond the technical skills defining my degree. Thank you.

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TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION………………………………………………………… 1 CHAPTER 2: LITERATURE REVIEW………………………………………………… 3 2.1 Placental Biology………………………………………………………………… 3 2.2 VEGFs and VEGFRs…………………………………………………………….. 3 2.3 FGFs and FGFRs………………………………………………………………… 5 2.4 VEGF and FGF Signaling – Akt and ERK……………………………………… 6 2.5 Alternative Splicing……………………………………………………………… 7 2.6 SR Proteins………………………………………………………………………. 8 2.7 SR Protein Kinases………………………………………………………………. 9

CHAPTER 3: CELL MODEL SELECTION, CHARACTERIZATION AND OPTIMIZATION…………………………………………………………………………... 11 3.1 Abstract…………………………………………………………………………. 11 3.2 Introduction…………………………………………………………………….. 11 3.3 Results…………………………………………………………………………… 12 3.3.1. Cell model Selection……………………………………………………... 12 3.3.2. Characterization and Development of Experimental Conditions……….. 14 HUVEC experimental lifespan is between passages 3 and 6……………… 14 Modified EGM1 yields variable expression of Flt1 variant mRNA………. 15 DMEM – cytotoxic pharmacological inhibition…………………………… 19 EGM2 - provides a suitable experimental environment in HUVECs……… 19 Experimental Conditions do not affect Cell Viability……………………… 21 Cell Cycle is affected by contact inhibition but not VEGF or FGF2 starvation…………………………………………………………………… 22 Real-Time qPCR Optimization……………………………………………. 24 3.4 Discussion……………………………………………………………………….. 27 3.5 Methods…………………………………………………………………………. 28 3.5.1 Materials………………………………………………………………….. 28 3.5.2 Cell Culturing…………………………………………………………….. 29 3.5.3 Cell Viability.…………………………………………………………….. 29 3.5.4 RNA isolation, Reverse Transcription, Endpoint PCR…………………… 29 3.5.5 SYBR Green and Taqman Real-Time qPCR……………………………... 30 3.5.6 DNA sequencing………………………………………………………….. 31 3.5.7 Annexin V / Propidium Iodide Staining for Flow Cytometry…………… 31 3.5.8 Cell Cycle analysis via Flow Cytometry………………………………… 32

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CHAPTER 4: VEGF AND FGF2 SIGNALS ARE TRANSDUCED THROUGH RELATED PATHWAYS TO DIFFERENTIALLY MODULATE FLT1 ALTERNATIVE SPLICING……………………………………………………………… 33 4A MANUSCRIPT, SUBMITTED FOR PUBLICATION……………………… 33 Abstract………………………………………………………………………… 34 Introduction…………………………………………………………………….. 35 Experimental Procedures……………………………………………………… 37 Materials………………………………………………………………… 37 Cell Culture……………………………………………………………… 37 RNA isolation, cDNA preparation and RT-PCR……………………….. 38 Statistical Analysis……………………………………………………… 38 Results………………………………………………………………………….. 39 rhVEGFA and rhFGF2 differentially modulate relative mRNA expression of Flt1 mRNA splice variants………………………………. 39 VEGF and FGF2 impact Flt1 pre-mRNA alternative splicing via related distinct signal transduction cascades……………………………………. 41 Figures………………………………………………………………………….. 43 Discussion………………………………………………………………………. 48 Literature Cited ……………………………………………………………….. 53

4B SUPPLEMENTARY RESULTS………………………………………………. 57 4B.1 Results……………………………………………………………………. 57 4B.1.1 Physiologically relevant levels of FGF2, but not VEGF, alter Flt1 variant splicing……………………………………………………… 57 4B.1.2 Cell Viability – Inhibitor treatments……………………………. 59 4B.2 Discussion………………………………………………………………… 61 4B.3 Methods…………………………………………………………………... 61

CHAPTER 5: IDENTIFICATION OF SR PROTEIN CANDIDATES……………….. 62 5.1 Abstract…………………………………………………………………….. 62 5.2 Introduction………………………………………………………………... 62 5.3 Results……………………………………………………………………… 63 5.3.1 Classical SR Proteins are present in HUVECs……………………. 63 5.3.2 Inhibition of SR protein kinases on Flt1 alternative splicing……… 65 5.3.3 Akt, but not ERK, is predicted to phosphorylate SR proteins…….. 68 5.3.4 Sites of predicted Akt phosphorylation in SR proteins are largely conserved………………………………………………………………….. 74 5.3.5 Predicted SR protein binding to Flt1 pre-mRNA………………….. 77 5.3.6 VEGF- and FGF2-signaling and SRSF2 and SRSF3 mRNA levels… 83 5.4 Discussion…………………………………………………………………... 87

5.5 Methods…………………………………………………………………….. 89 5.5.1 Materials…………………………………………………………… 89 5.5.2 Endpoint PCR and Real-Time qPCR………………………………. 89 5.5.3 Scansite……………………………………………………………... 91 5.5.4 Uniprot Species Alignment………………………………………… 91 5.5.5 SpliceAid 2…………………………………………………………. 91 5.5.6 Cell culture and treatments for phospho-proteins………………….. 91 5.5.7 SDS-PAGE and Immunoblotting…………………………………... 92 5.5.8 Statistical Analysis…………………………………………………. 91 CHAPTER 6: DISCUSSION……………………………………………………………… 93 CHAPTER 7: CONCLUSIONS…………………………………………………………... 98 REFERENCES……………………………………………………………………………... 100 APPENDICES……………………………………………………………………………… 114 A - AnnexinV/PI Flow Cytometry Raw Data…………………………………… 114 B - Cell Cycle Raw Flow Cytometry Data……………………………………… 126 C - Species Alignments for Predicted Akt Phosphorylation Sites in SR Proteins. 134 D - Raw SpliceAid 2 Data……………………………………………………….. 142 E – Materials: Vendors and Catalog Numbers………………………………….. 246

LIST OF FIGURES

# Title pg. 2.1 Flt1 Splice Variant Schematic 5 2.2 Growth Factor Signaling Pathways – Akt and ERK 7 3.1 Flt1 mRNA variants 14 3.2 Relative expression of Flt1 mRNA variants is stable in HUVECs through passage 7 15 3.3 BBE and FBS withdrawal affects HUVEC viability at 0.1% and 0.5%, respectively 16-17 3.4 Final concentrations of 0.1% BBE and 0.5% FBS in EGM1 do not affect cell viability compared to reference controls 18-19 3.5 Experimental Conditions 21 3.6 FGF2 and VEGF starvation do not induce apoptosis or cell death in HUVECs after 58h. 22 3.7 FGF2 and VEGF starvation and stimulation do not affect cell cycle in HUVECs at 0h or 48h. 23 3.8 Flt1 Splice Variant Schematic and qPCR Information 25 3.9 Efficiencies of simplex vs duplex RT-qPCR for Flt1 variant primer and probe pairs 26-27 3.10 Efficiency of SYBR Green RT-qPCR for sFlt1_v2 variant primers 31 4.1 Flt1 variants 43 4.2 VEGF-A alters Flt1 pre-mRNA splicing in a dose dependent manner while FGF2 shifts splicing toward sFlt1_v2 44-45 4.3 Combination treatments of rhVEGF-A and FGF 46 4.4 Inhibitor studies of Akt and ERK related pathways 47-48 4.5 FGF2, but not VEGF, induced Flt1 mRNA variant splice responses 58 4.6 Treatments with VEGF-E or PlGF did not uncover a VEGF-A signal or alter the FGF2-induced Flt1 splice variant pattern. 59 4.7 Effects of inhibition on HUVEC viability 60 5.1 Detection of SR proteins in HUVECs 64-65 5.2 Effects of SR protein kinase inhibition on Flt1 alternative splicing 67 5.3 Scansite Motif logos for Akt and Erk 69 5.4 Predicted Akt phosphorylation sites in SR proteins 69-74 5.5 SR protein maps to Flt1 pre-mRNA 79-83 5.6 FGF2- and VEGF- induced SRSF2 and SRSF3 mRNA relative expression in HUVECs at 48h 86 5.7 Regulation of SR protein mRNA by GFX-mediated VEGF stimulation and ERK- mediated FGF2 stimulation. 86 5.8 Efficiencies of simplex vs duplex RT-qPCR for SRSF2 and SRSF3 primer and probe pairs 90

LIST OF TABLES

# Title pg. List of known SR proteins examined for predicted Akt and ERK phosphorylation 5.1 68 motifs. 5.2 Conservation of predicted Akt phosphorylation sites in SR proteins. 75-77 Summary of SR protein predicted binding in portions of sFlt1_v1 and sFlt1_v2 5.3 83 pre-mRNA sequences. 5.4 Primers for SR Protein Endpoint PCR 90

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LIST OF ABBREVIATIONS

AS – alternative splicing; BBE – bovine brain extract; EGM – endothelial growth medium; ESE – exonic splice enhancer; FGF – fibroblast growth factor; Flt1 – Fms-related tyrosine kinase 1 (VEGFR1); hnRNPS – heterogeneous ribonucleoproteins; HUVECs - human umbilical vein endothelial cells; KDR – kinase insert domain receptor (VEGFR2); PE – preeclampsia; pEGM2 (endothelial growth medium without rhVEGF or rhFGF2); PI – propidium iodide; PlGF – placental growth factor; PMA (phorbol-12-myristate-13-acetate); sFlt1 – soluble Flt1 (Fms-like tyrosine kinase 1); SR proteins –serine/arginine rich proteins; VEGF – vascular endothelial growth factor

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CHAPTER 1: HYPOTHESIS and SPECIFIC AIMS

Vascular endothelial growth factor A (VEGF) is a crucial driver of angiogenesis, the formation of new blood vessels, via signals transduced through the tyrosine kinase receptors VEGFR1/Flt1 & VEGFR2/KDR. Bioavailability of VEGF is modulated by soluble forms of Flt1 (sFlt1_v1 & v2) that sequester VEGF in an anti-angiogenic fashion. Dysregulated production of these secreted variants is associated with vascular pathologies, including preeclampsia (PE), a pregnancy-related disorder responsible for 76,000 annual maternal and 500,000 fetal deaths worldwide [1]. PE and related hypertensive disorders of pregnancy impact 5-8% of all births in the United States while, in developing countries, more severe forms of preeclampsia are more prevalent [2, 3]. Marked by hypertension and proteinuria, PE is correlated with elevated VEGF and altered Flt1 mRNA splice variant ratios in the placenta [4-7]. The Ser/Thr kinase Akt has been implicated in analogous signal-induced alternative splicing events [8-13]. Akt is known to impact splicing by phosphorylating members of the Ser/Arg-rich (SR) protein family of splice regulators. We hypothesized that formation of alternative splice variants of Flt1 pre-mRNA is influenced by VEGF signal transduction via kinase Akt2 and its action on SR proteins. We tested this hypothesis using approaches divided into 2 aims:

Aim 1: Examine effects of activation of protein kinase signaling intermediates on relative abundance of Flt1 mRNA variants in cell culture. Flt1 splicing variant transcripts were examined via qRT-PCR in human umbilical vein endothelial cells (HUVECs). The influence of growth-factor stimulated signaling intermediates on Flt1 alternative splicing was investigated using pharmacological inhibition. Although our original hypothesis proposed exploration of VEGF signaling, over the course of our study we included investigation of fibroblast growth factor 2 (FGF2) impact on Flt1 alternative splicing.

Aim2: Identify specific SR proteins that may be potentially regulated by Akt in processing Flt1 pre-mRNA transcripts. We identified SR protein candidates that may be involved in growth-factor stimulated alterations of Flt1 splice variant expression levels using bioinformatic prediction tools. We examined growth factor-induced regulation of candidate SR proteins on the

1 level of transcriptional and. Involvement of SR protein kinases was examined via pharmacological inhibition.

CHAPTER 2: LITERATURE REVIEW

2.1 Placental Biology Proper placental vascularization is critical for the successful development of the fetus. The placenta is a unique vascular organ with two separate circulatory systems –maternal- placental and fetal-placental. The site of nutrient and oxygen exchange between the two systems takes place at the interface between the maternal decidua and fetal chorion in a space termed the intervillous space. Into this space extend embryonic villi containing capillary branches of umbilical vessels [14-16]. The villi are composed of three layers of cell groups – fetal vascular cells, stromal cells (e.g. mesenchymal cells), and a surface layer of cytotrophoblasts which give rise to syncytiotrophoblasts [17]. Maternal spiral arties empty into and perfuse the intervillous space, bathing the villi in nutrient-rich and oxygenated blood. In early pregnancy, invading cytotrophoblasts convert to an endothelial cell phenotype and replace the endothelial lining and vascular smooth muscle layer of the maternal spiral arteries [18-20], thus creating unrestricted maternal blood flow into the intervillous space. In addition to acting as a barrier between the fetal-maternal circulations and remodeling maternal spiral arteries, placental cytotrophoblasts secret a number of growth factors and hormones necessary for placental development, including VEGF, FGFs, and PlGF [21-26]. Autocrine and paracrine interaction of VEGF and its receptors (Flt1 & KDR) is critical to placental vasculogenesis and angiogenesis [27]. Improper remodeling of maternal spiral arteries leads to persistent hypoxia [28, 29] and is associated with preeclampsia [30-32]. In turn, hypoxia upregulates VEGF and Flt1 expression and downregulates PlGF expression. Increased expression of soluble Flt1 by cytotrophoblasts is highly correlated with the development of preeclampsia [4, 33-38]. In fact, soluble Flt1 and the sFlt/PlGF ratio are used as diagnostic markers of preeclampsia [39, 40]. It has recently been proposed that, rather than contributions of overall soluble Flt1 to preeclampsia, dysregulated Flt1 splice isoforms are the more likely contributors [4-6, 41].

2.2 VEGFs and VEGFRs The VEGF family of ligands consist of several isoforms – VEGF-A, VEGF-B, VEGF- VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF) – which are prominent molecules in angiogenesis and lymphangenesis. The VEGF isoforms bind with varying affinities

3 to VEGFR1 (Flt1), VEGFR2 (KDR) and VEGFR3. Of these, VEGF-A (hereto referred to as VEGF) is considered the major isoform involved in vasculogenesis (vessel formation from endothelial precursor differentiation) and angiogenesis (vessel growth or repair from existing vessels), thereby influencing development, maintenance and remodeling of the vasculature. New vessel growth is a highly complex process involving many factors; however, VEGF signaling is often the critical rate-limiting step [42]. Alternative splicing of VEGF gives rise to at least 9 isoforms with differing affinities for the extracellular matrix [43-46]. VEGF signals through two kinase receptors, Flt1 (VEGFR1) and KDR (VEGFR2). The kinase activity of Flt1 is about 10- to 20-fold less than that of KDR [47] and is unable to propagate a mitogenic signal except upon heterodimerization with KDR, which occurs in a limited fashion [48]. Consequently, Flt1 acts as a VEGF decoy by reducing its availability to KDR. Both KDR [49, 50] and Flt1 [4, 5, 51, 52] have alternatively spliced isoforms that are antiangiogenic in function. Flt1 alternative splicing yields 3 predominant isoforms: 1) Flt1 (NM002019) - a full-length trans-membrane protein kinase receptor, 2) sFlt1_v1 (U01134) – a soluble isoform [53] and 3) sFlt1_v2 (EU368830.1) - a recently described soluble isoform [4, 5, 51, 52] (Figure 2.1). The full-length Flt1 protein includes an intracellular kinase domain, a transmembrane domain, and an extracellular portion comprised of seven Ig-like domains [54, 55]. The soluble Flt1 (sFlt1) isoforms retain only the extracellular Ig-like domains and retain full VEGF binding capacity [47, 56]. The soluble forms of Flt1 perform an additional function as spatial regulators of emerging vessels sprouts [57, 58] via their ability to bind to heparan sulfate in the extracellular matrix [59]. Dysregulation of soluble Flt1 is believed to be one of the major contributing factors to the development of preeclampsia [4-6, 35, 41]. Specifically, the ratio of sFlt1_v2:sFlt1_v1 rises dramatically in preeclamptic placentas compared to those from normal pregnancy [5]. In support of this, a recent study demonstrated that adenoviral sFlt1 overexpression induced preeclamptic symptoms in mice, symptoms which were alleviated by reducing circulating sFlt1 levels with adenoviral VEGF delivery [36]. Moreover, anti-VEGF therapies induce PE-like symptoms [60, 61], further supporting the purported pathogenic role of dysregulated sFlt1 in PE. The factors that underlie Flt1 variant dysregulation in PE remain unknown. Hypoxia has been proposed as possible contributor to soluble Flt dysregulation, as it is known to increase sFlt1 expression [33, 35, 62- 66]. However, recent findings indicate that VEGF, itself, may influence sFlt1 expression [67, 68]. In particular, Fan et al. (2014) demonstrated that endometrial-specific overexpression of

VEGF induced increased placental sFlt1 (sFlt1_v1) expression which led to vascular defects and PE-like symptoms [68].

Figure 2.1. Flt1 Splice Variant Schematic. Flt1 pre-mRNA is alternatively spliced to yield a full-length transmembrane-spanning variant, Flt1, with 7 extracellular immunoglobulin-like domains. Two well characterized soluble variants, sFlt1_v1 and sFlt1_v2, are spliced prior to the transmembrane domain and consist of extracellular encoding exons. Consequently, sFlt1_v1 and sFlt1_v2 retain full VEGF-A binding capacity. Numbered boxes represent exons. Dashed boxes indicate unique variant sequences.

2.3 FGF2/FGFRs Although our hypothesis and aims did not include FGF2, through the course of our study we observed a significant impact of FGF2 signaling on Flt1 pre-mRNA splicing. Therefore, we include a review of FGF and its receptors here. The FGF family has a broad range of functions in tissue homeostasis and metabolism and play critical roles in virtually every step of development [69-71]. There are 18 true members of the FGF ligand family – FGF1-10 and FGF16-23, which function in a paracrine or endocrine fashion depending on their respective heparan sulfate affinities [72]. Although FGFs11-14 have high sequence homology with the FGF family, they do not activate FGFRs and are therefore not included in the FGF family [73]. (Beenken et al. provide a review of the physiological functions for each FGF {Beenken, 2009 #930}. FGFs signal through the tyrosine kinase receptor family of FGFRs. There are 4 FGFR genes (1-4) which are alternatively spliced to yield 7 predominant FGFRs.[74]. FGF2 (originally known as basic FGF), which binds FGFR1-4 [75], is well known for its role in angiogenesis and functions

5 synergistically with VEGF [76, 77]. Elevated levels of FGF2 have been found in serum of breast cancer patients [72, 78], as well as the maternal serum in hypertensive pregnancy [79]. Of note, FGF2 expression is elevated in cytotrophoblasts from preeclamptic placentas [80].

2.4 VEGF and FGF2 Signaling – Akt and ERK The VEGF signal is predominantly transduced through 3 pathways: 1) Raf1/MEK/ERK, 2) PI3K/Akt and 3) p38 MAP kinase. Utilizing similar signaling mechanisms, the FGF2 signal is largely transduced via 3 pathways: 1) Raf1/MEK/ERK, 2) PI3K/Akt and 3) PLCγ. Activation of MEK/ERK is generally associated with proliferation and gene transcription. The two isoforms of ERK (extracellular-regulated kinase/mitogen-activated kinase, MAPK) are serine/threonine kinases involved in a wide array of cellular functions via hundreds of substrates [81]. Activation of ERK1/2 occurs through MEK1/2, the only known kinase of ERK1/2 [82]. The core cascade unit of ERK1/2 activation, Raf1/MEK/ERK, can be activated via PKC or PKC-independent mechanisms [83-86], (Figure 2.2). As upstream initiators of ERK cascade through Raf1/ MEK/ERK, multiple isoforms and distinct components involved at each level lead to varied activation of the many ERK1/2 substrates [81, 82, 87, 88]. VEGF activates ERK via PKC [89- 92], whereas FGF may use either mechanism [71, 93]. VEGF and FGF2 both signal through PI3K/Akt which is associated with survival, permeability and migration [92] (Figure 2.2). The 3 related isoforms of Akt are serine/threonine kinases involved in various cellular functions. Akt1/2/3 is usually associated with PI3K activation, although PI3K-independent mechanisms have been described [94-99]. Recently, Akt has been described as kinase of SR proteins, thereby influencing alternative splicing [9, 12, 100]. Activation of p38-MAPK by VEGF leads to cytoskeletal regulation and migration [101]. FGF activation of PLCγ leads to intracellular release of Ca2+ and activation of PKC, which activates Raf, leading to ERK activation in a Ras- independent manner [83]. Considerable cross-talk between PI3K/Akt and MEK/ERK cascades is well documented (reviewed in [102, 103]). Externally-stimulated signaling mechanisms are complex and may affect the splicing of target genes in a variety of ways. A single pathway may affect a single gene or multiple genes. Additionally, the splice pattern of a single gene may be a product of one pathway or multiple pathways [104].

PI3 PLCγ PIP PIP K 2 2 Wortmannin GFX PIP DAG 3 MK220 PKC 6 PDK GRB2 Akt Raf1 SOS 1

Clks, MEK1/2 Ras TG003 SRPKs U0126 3

SRPIN SRp ERK1/2

Survival Permeability Pre-mRNA Transcription, splicing Migration Proliferation

Figure 2.2 VEGF and FGF2 Signaling Pathways – Akt and ERK. A basic schematic of two major growth factor signal transduction pathways are depicted. The PI3K/Akt pathway (purple pathway) is typically associated with survival, permeability, and migration [92]. Akt can lead to changes in pre- mRNA splicing by activating SR proteins directly or indirectly via SR protein kinases, Clk and SRPK [9, 12, 100]. Activation of RAf1/MEK/ERK (green pathways) can occur via PKC-dependent or – independent mechanisms [83-86]. ERK activation is typically associated with proliferation and gene expression. Inhibitors used to probe pathways are indicated in burgundy. Akt – protein kinase B ; Clk – cdc2-like kinase; DAG – diacyl glycerol; ERK – mitogen activated kinase; GRB2 - Growth factor receptor-bound protein 2; MEK – mitogen activated kinase kinase; PDK – pyruvate dehydrogenase kinase; PI3K (phosphoinositol 3 kinase); PIP - phosphatidylinositol 3-phosphate; PKC – protein kinase C; PLC – phospholipase C; Raf1 - proto-oncogene serine/threonine-protein kinase; SOS – son of sevenless; SRPK – SR protein kinase

2.5 Alternative Splicing The human genome contains about 20,000 protein-coding genes [105], from which an exponentially expansive proteome is coded. About 95 % of mammalian genes are alternatively

7 spliced (AS) to produce multiple proteins from one gene [106-108] that, in turn, can undergo multiple post-translational modifications that typically affect function. It is therefore crucial that factors driving AS must work concertedly and in a tightly regulated manner. Changes in any one contributing factor may lead to pathogenesis and are linked to various disease states (reviewed in

[109-113]). Due to association with specificity and severity of diseases, alternative splice patterns have been suggested as viable diagnostic biomarkers [114]. Further, a number of proposed mechanisms aimed at modulating various aspects of alternative splicing are being examined as potential drug targets [115-120]. At a fundamental level, splicing involves the removal of intronic sequences from precursor mRNA (pre-mRNA) by recognizing exon-flanking splice sites. Splicing takes place within a molecular complex of 4 small nuclear ribonucleoproteins (snRNPs), U1, U2, U4/U6 and U5, called the spliceosome, and about 50-100 additional splicing factors [121]. The decision by the spliceosome to use a variable splice site over a constitutive site determines whether a pre- mRNA will be alternatively spliced. The affinity of spliceosomal factors to splice sites is determined by consensus sequence agreement. The position of weak sites relative to strong sites allows for implementation of AS mechanisms such as exon inclusion/exclusion, use of alternative 5’ or 3’ splice sites, and intron retention. Ultimately, splice site selection is a result of a combination of factors, including splice site strength [122, 123], pre-mRNA secondary structure [124], factors related to transcription regulation such as transcription machinery, [125- 127] elongation rates [123, 128-133], presence of cis-regulatory elements [134, 135], and concentration and post-translational modification of RNA regulating proteins (trans-factors) [135-138]. Cis-elements include regulatory sequences found in either exons or introns that enhance (exon splice enhancers, ESEs, and intronic splice enhancers, ISEs) or silence splicing (exon splice silencers, ESSs, and intronic splice silencers, ISSs). The trans-acting factors that bind to these regions include two major splice regulating RNA-binding families, heterogeneous nuclear ribonucleoprotein (hnRNPS) and Serine/Arginine-rich proteins (SR proteins), as well as tissue-specific RNA-binding proteins such as NOVA [139] and FOX [140].

2.6 SR Proteins The families of hnRNPS and SR proteins often function antagonistically. While general splicing activities of hnRNPS and SR proteins do not seem to require high sequence specificity,

8 their functions as silencers and enhancers require binding to a specific target sequences [113]. HnRNPS are typically implicated in splice repression [141]. SR proteins, while often required for constitutive splicing, also influence alternative splicing by binding ESEs that lead to selection of suboptimal splice sites [142]. SR proteins may encourage intronic sequence inclusion by binding to nonconsensus splice sites [143] . Typically, binding of an SR protein within an exon promotes exon inclusion while binding to introns interferes with exon definition [144, 145]. Additionally, it has been shown that SR protein binding within an alternative exon promotes inclusion, whereas binding to flanking intronic sites promotes exon skipping [146, 147]. Individual SR proteins may have opposing effects on splicing [148, 149], may compete for ESEs, or may function cooperatively with other SR proteins [150]. SR proteins encourage alternative splicing in four ways – 1) interfering with hnRNP binding sites, 2) recruiting spliceosomal machinery to weak splice sites, 3) inhibiting bound hnRNP negative activity and 4) by creating a bridge to snRNPs via coactivator proteins, such as SR-like proteins that lack an RNA-recognition motif (RRM) [151]. There are currently 20 known SR proteins which, by definition, must contain at least one RRM and an RS domain and function in constitutive or alternative splicing [152]. Of these, 7 are considered “classical” SR proteins (SRSF1-7), which means they 1) share structural similarity, 2) have dual function in constitutive and alternative splicing, 3) contain a phosphoepitope recognized by mAb104; and 4) share puriﬁcation characteristics [138]. The RS domain, 50-100 residues [153], is highly subject to phosphorylation, which influences protein-protein interaction, localization and function [138, 153-155]. However, relative abundance of SR proteins may also contribute to alternative splicing in a tissue-specific or developmental manner [156, 157].

2.7 SR Protein Kinases There are a limited number of known SR protein kinases. The most well characterized SR proteins are SR protein kinase (SRPK), cyclin-dependent like kinase (Clk) and Akt. Isolated demonstrations of other kinases that have been shown to transfer phosphates to SR proteins in vitro include topoisomerase 1 [158], PKC [155] and dual specificity tyrosine phosphorylation- regulated kinases (DYRKs) [159]. The SRPK family, which is highly specific for SR proteins, is highly conserved and consists of 3 isoforms. SRPK1 is ubiquitously expressed, SRPK2 is expressed mostly in the

9 nervous system and SRPK3 is expressed in muscle cells [160, 161]. SRPKs are located in both the cytoplasm and nucleus [160] and transfer phosphates to SR proteins as a “polymerizing kinase” – transferring multiple phosphates without dissociation from the substrate after each round of the reaction [153]. The Clk family consists of 4 isoforms which are all dual-specificity kinases. Clks are localized to the nucleus where they co-localize with SR proteins [155, 162]. Like SRPKs, Clks act like polymerizing kinases [153]. It has been proposed that SRPKs and Clks synergistically phosphorylate SR proteins in a relay fashion [163], although distinct substrate specificity has been demonstrated [164]. Recently, Akt was demonstrated to both directly [9, 12, 100] and indirectly activate SR proteins by phosphorylating Clk [9] and SRPK [165, 166]. Akt is a Ser/Thr kinases that directs critical and diverse cellular functions, including growth, survival, proliferation and metabolism. Akt is largely activated via the PI3K pathway in response to growth factor stimulation; although other kinases have been demonstrated to activate Akt [94-99]. The Akt family has 3 closely related isoforms that have redundant and distinct functions [167-169]. Akt isoforms are largely expressed in a tissue-specific manner with Akt1 expression being ubiquitous [170-172], Akt2 expression in insulin-responsive tissues [170] and Akt3 expression mostly in the brain [173].

CHAPTER 3: CELL MODEL SELECTION, CHARACTERIZATION & OPTIMIZATION

3.1 Abstract Selection of a suitable cell model in which to conduct gene expression experiments ideally affords the benefits of physiological relevance, ease in obtaining and manipulation, as well as adequate expression of the targets of interest. Further, it is vital to establish a culture environment which is conducive to detecting changes in target gene expression in response to exogenous factors. From several considered possibilities, we selected human umbilical endothelial cells, HUVECs, as a fitting cell model in which to conduct our studies. We examined several experimental culture environments and ultimately developed a pre-treatment starvation period conducted in endothelial growth medium 2 (EGM2), from which FGF2 and VEGF are withdrawn for the duration of the experiment. We confirmed that this approach was not cytotoxic and, although it did not render cells quiescent, relative mRNA expression of Flt1 variants was reduced, creating a baseline from which response measurements to growth factor stimulation could be readily obtained.

3.2 Introduction Proper vascularization of the developing placenta is essential for fetal health and development. In normal pregnancy, maternal spiral arteries bathe embryonic villous trees with oxygen and nutrients [25]. Villous trees are finger-like projections that extend into a space between the fetal chorion and maternal decidua, into which the maternal spiral arteries empty. The outer layer of the villous trees is comprised of a syncytial layer of differentiated cytotrophoblasts, supplied from an underlying layer of cytotrophoblasts [25]. Cytotrophoblasts also convert from an epithelial to an endothelial phenotype and invade maternal spiral arteries [19], thereby replacing the endothelial lining and smooth muscle layer [25, 174]. In this manner, the maternal vessels offer low-resistance blood flow into the villous space, delivering a constant supply of nutrients and oxygen. Consequently, proper placental vascularization follows. In preeclampsia, for reasons unknown, the invasion of maternal spiral arteries is insufficient to support normal vascular development of the placenta [25, 30]. Upregulation of the soluble forms of Flt1 are correlated with preeclampsia [4, 33-38]. Levels of sFlt1 in the maternal serum drop

11 after delivery, as do the symptoms of preeclampsia. Consequently, placental production of sFlt1 was identified [175] and eventually further localized to cytotrophoblasts [64, 66]. Moreover, cytotrophoblasts have been identified as the predominate site of soluble Flt1 isoform dysregulation in preeclampsia [4-6, 41]. It has been proposed that soluble Flt1 dysregulation in cytotrophoblasts is responsible for abnormal placental vascularization in preeclampsia [35]. Factors influencing sFlt1 upregulation in the preeclamptic environment are unknown. One possibility is hypoxia, which is known to influence sFlt1 expression [33, 62, 64-66, 176]. Recently, VEGF has been demonstrated to upregulate sFlt1 production [67, 68]. In efforts to examine the impact of exogenous factors on Flt1 alternative splicing, we have established an effective cell model which allows us to measure growth-factor induced changes in Flt1 variant mRNA levels.

3.3 Results 3.3.1 Cell Model Selection To establish a cell model that was physiologically relevant, easy to handle and obtain, and also expressed our targets at reasonably detectable levels, we considered several possibilities. First, we explored primary cytotrophoblasts as a primary cell type. Cytotrophoblasts, and the derived syncytiotrophoblasts, are responsible for increased soluble Flt1 production in the preeclamptic placenta [4, 5, 66]. We arranged for collaboration with The Birthplace at Carillion in Radford, VA, to harvest human term placentas. We obtained a detailed protocol graciously given to us by Dr. Theresa Powell at The University of Texas Health Science Center. However, Dr. Powell advised us that successfully isolating primary cytotrophoblasts could take upwards of eight months, and may not ultimately be successful. Secondary to this, we initiated a potential collaboration with Drs Mark Longtine and Michael Nelson from University of Washington, who offered to provide primary cytotrophoblasts. Due to Dr’s Longtine and Nelson’s extensive experience with primary cytotrophoblasts, we understood that primary cytotrophoblasts are short lived in culture (72h maximum) and, to their knowledge, had not been successfully transfected. Given this, we looked to a line of immortalized cytotrophoblasts, HTR- SVneo, which were originally derived by Dr. Charles Graham at Queens University, ON, Canada, by transfecting cells from chorionic villi explants of human first-term placenta with the gene encoding for simian virus 40 large T antigen [177]. Dr. Graham graciously agreed to gift

12 these to us but explained that mRNA expression of soluble Flt1 was extremely low in these cells. Following this, we examined two choriocarcinoma lines, BeWo and JEG3, which are often used in lieu of or alongside cytotrophoblasts, despite the differences in genetic signatures [66, 178]. We examined mRNA relative expression of Flt1 variant targets via qPCR in BeWo and JEG3 cells. In both cells lines, mRNA expression was very low – posing potentially problematic expression in experiments that may reduce mRNA levels. At this juncture, we decided to use human umbilical vein endothelial cells (HUVECS). Although HUVECs are not trophoblasts, we noted that cytotrophoblasts which invade the maternal spiral arteries adopt an endothelial phenotype [19]. HUVECs were also attractive due to their embryonic origin, the ability to obtain them easily and from pooled donors, easy handling, and the adequate expression of Flt1 variant mRNA targets. We confirmed Flt1 variant mRNA expression in HUVECs via endpoint PCR (Figure 3.1a) using primers designed to target their unique sequences (Figure 3.1b). The bands were excised and sequence verified to reported sequences (Flt1 - NM002019, sFlt1_v1 - U01134, sFlt1_v2 - EU368830.1) via DNA sequencing.

A.) )

B.)

sFlt1_v1 sFlt1_v2 603 bp Flt1 603 bp

Expected Flt1 product –495bp Expected product – Expected sFlt1_v1 product – 586bp 635bp

Figure 3.1. Flt1 mRNA variants. Flt1 pre-mRNA is alternatively spliced to yield three predominant isoforms. Flt1 is a full-length, transmembrane tyrosine kinase receptor for VEGF. sFlt1_v1 and sFlt1_v2 are truncated, soluble isoforms of the Flt1 gene, which retain full-VEGF binding capacity and act as VEGF decoys. A) Schematic representation of Flt1 variant pre-mRNA. Numbered green boxes represent exons. Endpoint PCR primers (blue triangles) were designed to detect the unique sequences of each variant. B) Endpoint PCR confirmed presence of the three targeted Flt1 variants. Identities were confirmed via DNA sequencing.

3.3.2 Characterization and Development of Experimental Conditions HUVEC experimental lifespan is between passages 3 and 6 Next, we characterized the working HUVEC lifespan in culture, as it pertained to the stability of Flt1 variant mRNA. We measured the relative mRNA expression of Flt1 variants via qPCR over a period of eight passages (Figure 3.2). Flt1 variant relative expression remained stable through passage 7. Henceforth, we conducted all experiments in passages 4, 5 and 6.

v1:Flt1 0.1 v2:Flt1 v1:v2 RQ RQ Ratios 0.01 p2 p3 p4 p5 p6 p7 p8

HUVEC Passage Number

Figure 3.2. Relative expression of Flt1 mRNA variants is stable in HUVECs through p7. HUVECs were maintained in EGM1 and harvested at each passage for 8 passages. Flt1 mRNA targets were measured via SYBR Green qPCR and were normalized to p2 samples. Relative quantitation (RQ) values are expressed as variant ratios, rather than normalization to an internal housekeeping gene. As conformational preliminary data, biological replicates are not represented.

Modified EGM1 yields variable expression of Flt1 variant mRNA Next, we established non-cytotoxic culture conditions that yielded measurable responses of Flt1 mRNA variants to VEFG and FGF2 treatments in endothelial growth medium 1 (EGM1). EGM1 (Lonza) consists of basal medium with gentamicin, hydrocortisone, ascorbic acid, rhEGF, FBS and bovine brain extract (BBE). In efforts to create a baseline from which we could measure a response to VEGF treatment, we attempted to reduce the amount of FBS (2%) and BBE (0.4%) in experimental conditions while balancing cell toxicity. We incrementally reduced either FBS or BBE while holding the other constant. We assessed cell viability objectively via trypan blue cell counts (Figure 3.3a) as well as through subjective visual assessments (Figure 3.3b), using 2% FBS and 0.4% BBE treatments as “100% cell viability” control reference. Between 0.1% – 0.04 % BBE there was a drop in cell viability (85.9% and 54.2% viable, respectively). A similar drop was seen with FBS reduction, between 0.5 – 0.2% (90.2% and 40.7% viable, respectively). We then applied the cytotoxic cutoffs of 0.1% BBE and 0.5% FBS to conduct a second trial using 1) 0.1% BBE with varied levels of FBS and 2) 0.5% FBS with varied levels of BBE (Figure 3.4) to assess a combined reduction of FBS and BBE levels. In general, cell viability counts were more variable than in the first trial. Despite the variability, we concluded that cell counts and visual assessments indicated that combination of 0.5% FBS and 0.1% BBE in experimental medium did not further reduce cell viability. We then performed

preliminary treatments on cells with VEGF, PMA (Phorbol-12-Myristate-13-Acetate), Cobalt Chloride (a hypoxia mimetic), and insulin. We measured Flt1 mRNA relative expression via SYBR Green qPCR. Results were highly variable and extremely inconsistent. We reasoned that factors present in the BBE made this an unsuitable medium in which to conduct our studies.

100 100 A.) B.)

90 90

80 80 70 70 60 60 50 50 40 40

30 Cell Cell Count,% Control Cell Cell count, % control 30 20 20 10 10 0 0

% BBE % FBS C.)

D.)

Figure 3.3. BBE and FBS withdrawal affects HUVEC viability at 0.1% and 0.5%, respectively. HUVECs were maintained in EGM1. BBE or FBS were withdrawn to assess impact on cell viability via trypan blue staining. Cell counts are made relative to counts of cells in complete medium (with 0.4% BBE and 2.0% FBS, which were 80% viable) are referenced as 100% viable. A) All factors in EGM1 remained constant while HUVECs received varied amounts of BBE (0.4% = complete EGM1) for 36h. Cell viability dropped sharply between 0.1% and 0.4%. B) All factors in EGM1 remained constant while HUVECs received varied amounts of FBS (2.0% = complete EGM1) for 36h. Cell viability was variable but dropped sharply between 0.5% and 0.0%. C, D) Light microscope images (20x) of HUVECs under varied conditions of BBE (C) and FBS (D). Visual image assessments support trypan blue assessed cell viability. BBE – Bovine Brain Extract, FBS – Fetal Bovine Serum

B.) A.) 140 120

120 100

100 80 80 60 60 40

Cell Cell Count,% control Cell Cell Count,control % 20 20

0 0 0.4 0.2 0.15 0.1 0.075 0 2 1 0.75 0.5 0.25 0 % BBE (with 0.5% FBS) % FBS (with 0.1% BBE)

C.)

D.)

Figure 3.4. Final concentrations of 0.1% BBE and 0.5% FBS in EGM1 do not affect cell viability compared to reference controls. HUVECs were maintained in EGM1. BBE or FBS were withdrawn to assess impact on cell viability via trypan blue staining. In general, cell viability was variable. A) All factors in EGM1 remained constant with FBS at 0.5% while HUVECs received varied amounts of BBE for 36h. Cell viability dropped sharply between 0.075% and 0%. Cell counts are made relative to counts of cells in 0.4% BBE with 0.5% FBS (which were 78% viable) and are referenced as 100% viable. B) All factors in EGM1 remained constant with BBE at 0.1% while HUVECs received varied amounts of FBS for 36h. Cell viability dropped sharply between 0.5% and 0%. Cell counts are made relative to counts of cells in 0.1% BBE with 2.0 % FBS (which were 77% viable) and are referenced as 100% viable. C, D) Light microscope images (20x) of HUVECs under varied conditions of BBE (C) and FBS (D). Visual images reflect trypan blue assessed cell viability. BBE – Bovine Brain Extract, FBS – Fetal Bovine Serum

DMEM – cytotoxic pharmacological inhibition We then examined the use of low-glucose DMEM (10% FBS + 50ug/mL gentamycin) for our experimental conditions, as this had previously been used successfully in migration studies in our laboratory. Although the cells were stressed (as assessed visually), Flt1 variant mRNA relative expression levels were consistent when treated with VEGF. However, initiation of inhibitor studies resulted in cell death by 48h. Therefore, DMEM was not a suitable medium in which to carry out our experiments.

EGM2 – provides a suitable experimental environment in HUVECs

We changed from HUVECS grown/maintained in EGM1 to those grown/maintained in EGM2 – a more defined endothelial growth medium. EGM2 (Lonza) lacks BBE which is replaced by aliquots of rhEGF, rhIGF, rhFGF, and rhVEGF. Since we were interested in establishing a baseline from which we could measure a response to VEGF, (and were unable to arrest HUVECs via serum starvation), we removed VEGF from the medium and began to conduct preliminary experiments to assess various factors, including length of treatments, withdrawal impact of the remaining growth factors on Flt1 variant mRNA relative expression, and periods of pre-treatment growth factor starvation. We observed that, by withdrawing FGF2 and VEGF from the medium (denoted pEGM2) for 10h prior to treatments, we were able “quiet” Flt1 variant relative mRNA expression compared to those maintained in EGM2 (Figure 3.5a). Specifically, for 0h EGM2 vs pEGM2 samples compared to 10h, sFlt1_v1 increased 1.65 ± 0.05 fold and 1.08 ± 0.06 fold, respectively; sFlt1_v2 increased 1.78 ± 0.04 fold and 1.27 ± 0.05 fold, respectively; Flt1 increased 1.81 ± 0.04 fold and 1.22 ± 0.04 fold, respectively. Consequently, we established a pre-treatment “starvation” period of 10h followed by treatment at 0h. We determined 48h optimal for harvest, as changes in sFlt1_v2 variant expression were more pronounced at this time, compared to 24h (see Figure 4.5). Cultures remained in pEGM2 during the treatment period (Figure 3.5b). With this approach, we were able to create a baseline from which Flt1 splice variant mRNA relative expression changes could be readily detected.

A.) 2.0

1.5

sV1

RQ sV2

1.0 Flt1

0.5 EGM2 pEGM2

B.)

Figure 3.5. Experimental Conditions. We empirically determined HUVEC experimental culture conditions (modified medium, starvation factors, length of treatments) that created a baseline from which changes in Flt1 variant mRNA relative expression levels could be detected. A.) HUVECs were placed in fresh EGM2 or pEGM2 (EGM2 without rhFGF2 and rhVEGF) at “-10h” for 10h as a starvation period prior to experiments. Flt1 variant mRNA relative expression at 0h was measured via qPCR and referenced to -10h, using HPRT as a normalizing gene. Expression levels of each Flt1 variant was reduced in the pEGM2 sample compared to the EGM2 sample. Each biological sample was run in duplicate. Error bars are represented at ± SEM; n=3. B.) Experimental Timeline: at 10h prior to growth factor treatments (-10h), conditioned EGM2 is replaced by pEGM2. At 0h, growth factors treatments are initiated. Samples are harvested at 48h post treatments.

Experimental conditions do not affect cell viability Annexin V is a calcium dependent phospholipid-binding protein which binds to phosphotidylserine that has flipped to the extracellular membrane. Fluorescently labeled Annexin V allows for measurement of cells in early apoptosis. In late stage apoptosis, loss of membrane integrity allows for uptake of propidium iodide (PI). Annexin V/ PI staining was performed to assess viability impact of culture conditions, in terms of apoptosis and cell death (Figure 3.6). Cells cultured in EGM2 were placed in fresh EGM2 or pEGM2 at -10h. At 0h, cells in EGM2 and pEGM2 received either 0.1% DMSO, nothing or, for a portion of those in pEGM2, received basal amounts of VEGF and FGF2 (equaling complete EGM2). Cells were stained with PacificBlue and PI and were analyzed via flow cytometry. Cells which were in EGM2 and pEGM2 for 58h were 85.6% and 83.3% viable, 9.35% and 11.1 % dead, and 1.59%

21 and 1.10% apoptotic, respectively. (Raw Flow Cytometry data can be found in Appendix A.) There were no significant changes observed in cells that received DMSO or VEGF+FGF2 at 0h. These data indicate that 58h of VEGF and FGF2 starvation, with or without DMSO, does not induce apoptosis or cell death.

100

50 Live Cells

% Cells % Early Apoptotic Cells 40 Dead Cells 30

Figure 3.6. FGF2 and VEGF starvation do not induce apoptosis or cell death in HUVECs after 58h. HUVECs were cultured in either EGM2 or pEGM2 for 58 hours – the length of time corresponding to experiments for mRNA analysis – or pEGM2 for 10h. At 0h, cells received no treatment or VEGF (2ng/mL) + FGF2 (4n/mL) or 0.1% DMSO for 48h. Harvested adherent and floating cells were stained with PacificBlue and Propidium Iodide (PI) and analyzed via flow cytometry. Raw flow cytometry data may be found in Appendix A.

Cell cycle is affected by contact inhibition but not VEGF or FGF2 starvation As HUVECs cannot be arrested or synchronized through serum starvation, we examined the effect of VEGF and FGF2 starvation on cell cycle (Figure 3.7). Cells were fixed in ethanol, stained with PI, and cell cycle was analyzed via flow cytometry to assess cell cycle. (Raw Cell Cycle Flow Cytometry Data can be found in Appendix B). Prior to -10h, cells were maintained in EGM2. At -10h, cells either received fresh EGM2 or pEGM2. Cells analyzed at -10h were 48.13% ± 0.23% in G1 phase, 26.23% ± 1.63% in S phase and 26.38% ± 1.48% in G2 phase. Cells analyzed at 0h demonstrated no significant changes in cell cycles for either EGM2 or pEGM2 samples. Specifically, for EGM2 samples, 48.10% ± 4.10% were in G1 phase, 28.25% ± 1.45% were in S phase and 24.50% ± 3.40% were in G2 phase. For samples in pEGM2, 53.90%

± 1.90% were in G1 phase, 23.30% ± 1.20% were in S phase and 23.80% ± 3.0% were in G2 phase. At 0h, cells remained in EGM2, pEGM2 or received growth factor treatments. Cells analyzed from EGM2 and pEGM2 at 48h demonstrated an increase in G1 phase cells (79.55% ± 2.15% and 80.30% ± 1.20%, respectively) with corresponding decreases in S phase (10.21% ± 1.20% and 9.72% ± 2.58%, respectively) and G2 phase (11.13% ± 1.27 and 10.38% ± 1.23%, respectively) (Figure 3.7a). This pattern shift in cell cycles was mimicked in growth factor treated samples at 48h, regardless of treatment type or medium (Figure 3.7b). We concluded that neither VEGF/ FGF2 starvation nor stimulation affects cell cycle within our experimental parameters. However, by 48h, when cells are confluent, proliferation is reduced. We reason this is due to contact inhibition.

A.) B.) 90 90 EGM2 pEGM2

80 80

70 70

60 60

50 50

40 40

G1 Cell Cell Cycle % 30 G1 30

Cell Cell Cycle % S S 20 20 G2 G2 10 10

rhVEGF(50n/mL) rhFGF2(4ng/mL) pEGM2 rhVEGF(50ng/mL) rhFGF2(4ng/mL) rhVEGF(50ng/mL) + FGF2 rhVEGF(2ng/mL) + FGF2

0 EGM2

-10h 0h,EGM2 0h,pEGM2 48h,EGM2 48h,pEGM2

(4ng/mL) (4ng/mL)

Time course treatments Treatments

Figure 3.7. FGF2 and VEGF starvation and stimulation do not affect cell cycle in HUVECs at 0h or 48h. Cells were fixed in ethanol and stained in PI to assess cell cycle via flow cytometry. A) Cells were placed in either EGM2 or pEGM2 at -10h. At time 0h, corresponding to treatment initiation, cell cycle was assessed. There was little change in cell cycle from -10h to 0h. However, at 48h, proliferation decreased as evidenced by the decrease in S and G2 phases and increase in G1 phase. B) Cells were treated with various combinations of FGF2 and VEGF to assess their impact on cell cycle. No meaningful changes were observed regardless of treatment. Error bars are represented as ± SEM; n=2.

Real-Time qPCR Optimization We decided to further refine the SYBR Green qPCR assay by designing primers and probes for use with Taqman reagents. We targeted sequences unique to each Flt1 variant and designed probes to cross exon/exon boundaries where possible, to reduce genomic DNA amplification. We also added a “total Flt1” target which would capture all three Flt1 variants (Figure 3.8). In addition, we switched from 18S to HPRT (hypoxanthine phosphoribosyltransferase) as a housekeeping gene which is reported to be very stable in HUVECs [179]. Primers and probes for Flt1, total Flt1 and HPRT were obtained as predesigned assay mixes from ABI. We custom designed the primers and probes for sFlt1_v1 and sFlt1_v2. With the intention of detecting targets via multiplex qPCR we examined all Flt1 variant mRNA targets in simplex (one target per well) and in various duplex (two targets per well) combinations. Similar to primer validation, it was necessary to combine target pairs that do not affect the amplification efficiency of either target, compared to each target alone. Following examination of several possible combinations, we paired sFlt1_v1 with HRPT which yielded efficiencies of 99% and 100%, respectively, in both simplex and duplex qPCR – resulting in no changes in efficiency (Figure 3.9a). We paired sFlt_v2 with Flt1 (Figure 3.9b). SFlt1_v2 amplification was 100% efficient in both simplex and duplex qPCR. We observed 91% efficiency in Flt1 when duplexed, compared to 100% efficiency in simplex. However, even as this resulted in 9% efficiency reduction, it falls within the accepted range of 90% – 100%. All other possible combinations were less favorable and we considered this a reliable pairing. We also validated the pairing of total Flt1 with KDR (Figure 3.9c); however, all total Flt1 qPCRs were ultimately run in simplex. In summary, we validated target pairs for duplex Taqman Real- Time qPCR which demonstrated high amplification efficiencies. From this, we were able to confidently analyze the impact of our experiments on Flt1 variant mRNA relative expression.

Figure 3.8. Flt1 Splice Variant Schematic and Information. Flt1 pre-mRNA is alternatively spliced to yield a full-length transmembrane-spanning variant, Flt1, with 7 extracellular immunoglobulin-like domains. Two well characterized soluble variants, sFlt1_v1 and sFlt1_v2, are spliced prior to the transmembrane domain and consist of extracellular encoding exons. Consequently, sFlt1_v1 and sFlt1_v2 retain full VEGF-A binding capacity. In addition to these 3 Flt1 variants, Real-Time qPCR targets include “total Flt1”, which captures all three variants to measure collective relative expression levels. Numbered boxes represent exons. Taqman qPCR primers and MGB probes are indicated by arrows and rectangles, respectively. Dashed boxes indicate unique variant sequences.

A.) 40 38 36 Slopes = Efficiency sV2-sim: -3.00 = 100% 34 Flt1-sim: -3.27 = 100%

32 sV2-dup: -3.33 = 100%

30 Flt-dup: -3.55 = 91% Ct 28 sV2-simplex 26 Flt1-simplex 24 sV2-duplex 22 Flt1-duplex 20 0.6 1.6 2.6 3.6 4.6 RNA, log equivalents (pg)

B.) 40 38 Slopes = Efficiency sV1-sim: -3.35 = 99% 36 hprt-sim: -3.27 = 100% 34 sV1-dup: -3.35 = 99% hprt-dup: -3.28 = 100% 32

30 sV1-simplex

Ct 28 hprt-simplex

26 sV1-duplex 24 hprt-duplex 22 20 0.6 1.6 2.6 3.6 4.6 RNA, log equivilants (pg)

C.) 38 36 Slopes = Efficiency tFlt1-sim: -2.45 = >100% 34 KDR-sim: -2.38 = >100% 32 tFlt1-dup: -2.43 = >100% KDRt-dup: -2.33 = >100%

Ct tFlt1 - Simplex 28 tFlt 1 - Duplex 26 24 KDR - Simplex 22 KDR- Duplex 20 0.6 1.6 2.6 3.6 4.6 RNA, log equivilants (pg)

Figure 3.9. Efficiencies of simplex vs duplex RT-qPCR for Flt1 variant primer and probe pairs. Amplification efficiencies were compared using a single cDNA target per qPCR well (simplex) or two mRNA targets per well (duplex). The following pairs were ultimately chosen based on high efficiency rates in both

simplex and duplex qPCR. A) Flt1 and sFlt1_v2 efficiencies, B) HPRT (housekeeping gene) and sFlt1_v2 efficiencies, C) Total Flt1 (pan Flt1) and KDR efficiencies.

3.4 Discussion We began this project by examining several potential cell models in which to carry out experiments. In preeclampsia, cytotrophoblasts and syncytiotrophoblasts are believed to be responsible for soluble Flt1 dysregulation. Primary cytotrophoblasts in cell culture, however, are very difficult to isolate, have a very short lifespan in culture and are difficult to manipulate. Immortalized cytotrophoblasts created by Dr. Charles Graham at Queens University, ON, Canada, HTR-SVneo, as he explained, revealed very low expression of our desired targets. Examination of two choriocarcinoma lines, BeWo and JEG3, by measuring Flt1 variant targets via SYBR Green qPCR, resulted in very low mRNA detection levels. Finally, we examined human umbilical vein endothelial cells, HUVECs, which were attractive for several reasons - embryonic derivation, ability to be transfected, primary as opposed to immortalized cells, ease of obtaining, ability to pool donors and acceptable detection of Flt1 variant targets. However, as Flt1 variant expression is likely cell-specific, future validation of reported findings in a trophoblast cell type would be informative. Having identified a suitable cell model, we endeavored to establish a baseline from which we could observe changes in our desired targets. Ordinarily, serum starvation is the preferred method used to arrest and synchronize cells – an environment not favorable for HUVEC survival. Consequently, we started by using a basal endothelial medium that is supplemented with bovine brain extract (BBE) and FBS, along with other basic factors, (EGM1). As BBE and, to a lesser extent FBS, contain many growth factors, we systematically reduced the concentration of both BBE and FBS in EGM1 in order to reduce growth factor-induced signaling. However, as a relatively undefined medium, it resulted in inconsistent and variable qPCR measurements of Flt1 variant targets. We then examined low-glucose DMEM that, when combined with pharmacological inhibition, resulted in cell death. Finally, we used EGM2, endothelial growth medium 2, in which BBE is replaced by a defined set of growth factors. In examining the effect of withdrawal of these growth factors from the experimental medium, we observed a consistent and strong response of Flt1 variant mRNA expression to FGF2 presence. We found that by withdrawing FGF2 and VEGF from the medium 10h prior to treatments for the duration of

27 experiments, we were able to reduce relative mRNA expression of Flt1 variants, compared to complete EGM2. The observed reduction of Flt1 mRNA expression in pEGM2 vs EGM2 was not due to altered cell cycle, as evidence by cell cycle analysis via flow cytometry, but was instead due to withdrawal of VEGF and FGF2. However, at 48h, G1 phase cells increased and cells in S and G1 phases decreased, likely due to contact inhibition. As HUVECs require various factors to survive, we also determined that withdrawal FGF2 and VEGF from the experimental medium was not toxic to cells as assessed via Annexin V / PI staining and subsequent analysis via flow cytometry. Additionally, we refined our qPCR assay by developing and validating primer and probe pairs for duplex Taqman qPCR. In summary, we established a viable and nontoxic cell model and culture conditions, in which to readily detect Flt1 variant mRNA relative expression. We developed a reliable multiplex qPCR assay designed to measure unique Flt1 variant sequence targets. Of note, given the unexpected Flt1 variant mRNA expression response to FGF2, we decided to investigate the effect of FGF2 on Flt1 variant mRNA expression, in addition to planned VEGF experiments.

3.5 Methods 3.5.1 Materials JEG3 cells (HTB-36), BeWo cells (CCL-98) and Eagle’s Modified Essential Medium (EMEM) and F-12K were obtained from ATCC. Fetal Bovine Serum (FBS) was from Gibco. Human umbilical Vein Endothelial Cells, HUVECs (catalog no. CC-2519 & CC-2519A),

Endothelial Growth Medium 1, EGM1, and EGM2 BulletKit were obtained from Lonza. RNeasy Mini Kit was from Qiagen. T75 flasks and 60mm tissue culture treated plates were from Corning. Trypsin/EDTA, 0.25%Trypsin/21mM EDTA in HBSS, was from Cellgro. The High Capacity cDNA RT Kit was from Applied Biosystems. Taq PCR Master Mix for endpoint PCR was from Qiagen. Dulbecco’s Phosphate Buffered Saline, DPBS, was from Cellgro. Agarose powder for gels was from Sigma. Qiaex II Gel Extraction Kit was from Qiagen. SYBR Green and qPCR reagents targeting 18s were from Applied Biosystems. Gentamycin was from CellGro. Taqman Gene Expression Master Mix, Gene Expression Assay Mixes for pan/total Flt1 (catalog no Hs01052961) and full-length Flt1 (catalog no Hs1052944) and custom probes were from Applied Biosystems. AnnexinV/PI staining Binding Buffer and PacBlue Annexin V conjugate were from Life Technologies. Propidium iodide was from Thermo Fisher.

3.5.2 Cell Culture JEG and BeWo cells were grown and maintained in EMEM and F-12K, respectively, with 10% FBS. HUVECs, pooled from three donors, were maintained in EGM1 (2% FBS), low- glucose DMEM (10% FBS) or EGM2 (2% FBS). All cultures were incubated at 37C with 5% CO2 in T75 flasks and passaged to 60mm plates for RNA analysis. Subcultivations were at 1:5 (JEG3), and 1:3 (BeWo), and 2500 cells/cm2 (HUVECs), by rinsing with DPBS followed by incubation with 0.25% Trypsin/21mM EDTA for 5min at room temperature. HUVECs were seeded at 7500 cells/cm2 for experiments.

3.5.3 Cell Viability Cell viability was assessed by trypan blue cell staining. Cells were detached by incubation with 0.25% Trypsin/21mM EDTA. After 5m, complete EGM1 was added to the Trypsin/EDTA at a 1:1 volume and transferred to 14mL conical tube. The plates were rinsed with DPBS, and added to the appropriate tube. Cells were spun down at 300 x g for 5min and resuspended in complete EGM1. Trypan blue was added to cells at 1:1. The total numbers of cells were counted, as were the numbers of viable cells and dead cells. The number of viable cells in a sample was divided by the number of total cells for that sample to yield a viability fraction. The viability fraction for each sample was divided by the viability fraction of the control reference sample, multiplied by 100 to yield a percent viability.

3.5.4 RNA isolation, Reverse Transcription, Endpoint PCR Cells were lysed and total RNA was isolated using Qiagen’s RNeasy Mini Kit according to manufacturer’s instructions. RNA was reverse transcribed using ABI’s High Capacity cDNA RT Kit with 2ug total RNA according to manufacturer’s instructions. cDNAs were amplified using Taq PCR Master Mix (Qiagen) on a Thermocycler PCR (Hybaid). Endpoint PCR was also run on the Thermocycler PCR (Hybaid), with the following parameters: Stage 1, 1 cycle (94C, 2m; 55C, 1m; 72C, 1m); Stage 2, 28 cycles (94C, 1m; 55C, 1m; 72C, 2m); Stage 3, 1 cycle (94C, 1m; 55C. 1m, 72C, 10m). Primers were designed to target unique sequences for: Flt1 F: GACGGAAGGAGAGGACCTGAA-3’ (BH- 658) R: TTGCAGTGATAGACACCTTCATC-3’ (BH- 662)

29 sFlt1_v1 F: CACCTTGGTTGTGGCTGACTC- 3’ (BH-663) R: TCTCCTCCGAGCCTGAAAGTT-3’ (BH-659) sFlt1_v2 F: GACGGAAGGAGAGGACCTGAA-3’ (BH-658) R: CCCGGCCATTTGTTATTGTTA-3’ (BH-651).

3.5.5 SYBR Green and Taqman Real-Time qPCR SYBR Green Real Time qPCR was run on Applied Biosystems 7300 Real-Time PCR System using SYBR Green and the following parameters: Stage 1, 1 cycle (50C, 2m); Stage 2, 1 cycle (95C, 10m); Stage 3, 40 cycles (95C, 15s; 60C 1m); Stage 4 (dissociation), 1 cycle (95C, 15s; 60C, 20s; 95C, 15s; 60C, 15s. Samples were normalized to 18s as a housekeeping gene. The primers designed for SYBR Green RT-PCR were: Flt1 F: TATGCCTGCAGAGCCAGGAA -3’ (BH- 386) R: CTGAGGTTTCGCAGGAGGTATG -3’ (BH- 388) sFlt1_v1 F: TATGCCTGCAGAGCCAGGAA - 3’ (BH-386) R: TTTGGAGATCCGAGAGAAAACAG -3’ (BH-387) sFlt1_v2 F: TCCTGCGAAACCTCAGTGAT-3’ (BH-671) R: ACGATGACGATGGTGACGTT -3’ (BH-672). Primers were designed and validated previously, with the exception of sFlt1_v2. We performed 5-fold serial dilutions of cDNA derived from 1ug RNA and assessed the efficiency at 97% from a slope of -3.40 (Figure 3.10). Taqman Real Time qPCR was run on Applied Biosystem’s StepOnePlus Real-Time System, using Taqman Gene Expression Master Mix using the following parameters: Stage 1, 1 cycle (50C, 2m; 95C, 10m); Stage 2, 40 cycles (95C, 15s; 60C, 1m). Predesigned ABI assay mixes were used for full-length Flt1 and pan/total Flt1. We designed custom probes and primers for sFlt_v1 (probe 5’ - FAM-ACAATCAGAGGTGAGCACT-MGBNFQ – 3’, and primers F: TGCCTGCAGAGCCAGGA–3’, R: GTGGTACAATCATTCCTTGTGCTTT-3’) and for sFlt1_v2 ( probe 5’ - VIC-AAGAGCCTGAACTGTATACA-MGBNFQ – 3’ and primers F: CGAGCCTCAGATCACTTGGTTT-3’, R: GATGACGATGGTGACGTTGATG-3’); (MGB – minor groove binder; NFG – nonfluorescent quencher).

33 32 31 Slope = -3.396

Ct 29 28 27 26 25 2.51 3.2 3.9 4.6 Log RNA (pg)

Figure 3.10. Efficiency of SYBR Green RT-qPCR for sFlt1_v2 variant primers. Amplification efficiencies assessed using 5-fold serial dilutions of HUVEC cDNA. A slope of -3.396 corresponds to 97% amplification efficiency.

3.5.6 DNA sequencing To confirm identify of amplified products, PCR reaction mixes were fractionated on a 2% agarose gel with Tris/Borate/EDTA (TBE) buffer, the appropriate bands excised, and cDNA extracted using Qiagen’s Qiaex II Gel Extraction Kit, according to manufacturer’s instructions. 5pmol/ul primers and 10ng/ul PCR products were submitted for DNA sequencing. Results were aligned using SeqMan Assembly in Lasergene.

3.5.7 Annexin V / Propidium Iodide Staining for Flow Cytometry Condition medium was removed from cells and collected in designated 14mL conical Falcon tubes. Cells were rinsed with DPBS which was added to the appropriate Falcon tube. Adherent cells were then incubated with 1:1 dilution of 0.25% Trypsin/21mM EDTA to DPBS at room temperature for 5min, and dislodged by tapping. EGM2 was added to the plate and pooled to the appropriate Falcon tube. The plates were rinsed with DPBS, which was also pooled. The cells were spun down at 350 x g for 5m at 4C. The supernatant was aspirated and the cells washed in ice-cold DPBS. Cells were kept on ice for the remainder of the procedure. Pellets were resuspended in cold 100uL 1x Binding Buffer (to which 2uL 100 ug/mL PI and 5ul PacificBlue had already been added). Three controls were prepared, which contained either 1x Binding Buffer alone, 1x Binding Buffer + PacificBlue, or 1x Binding Buffer + PI. Samples were analyzed immediately and were acquired on a BD FACSAria I (BD Biosciences, San Jose,

CA). The gates were set to save 10,000 single cells per sample. Data was analyzed using FlowJo v10 software (TreeStar, Inc.).

3.5.8 Cell Cycle analysis via Flow Cytometry Condition medium was removed from cells and discarded. Cells were rinsed with DPBS and then incubated with 1:1 dilution of 0.25% Trypsin/21mM EDTA to DPBS at room temperature for 5min, and dislodged by tapping and quenched with EGM2. Cells were transferred to a 14mL conical Falcon tube, the plates were rinsed with DPBS, which was added to the falcon tube. The cells were spun down at 350 x g for 5min at 4C. The supernatant was aspirated and the cells washed in ice-cold DPBS. To the pellet, 1ml cold 70% Ethanol was added dropwise, while gently vortexing, followed by a 30m incubation at 4C. Tubes were sealed and stored at -20C for two weeks. When cells were thawed they were washed twice in DPBS (centrifugation at 850 x g). Directly to the pellet, 50uL of 100ug/mL RNase was added and mixed well. To this, 200uL of 50ug/mL PI was added, followed by overnight incubation at 4C. Samples were acquired on a BD FACSCalibur (BD Biosciences, San Jose, CA). The gates were set to save 10,000 single cells per sample. Data was analyzed using FlowJo v7 software (TreeStar, Inc.).

CHAPTER 4: VEGF AND FGF2 SIGNALS ARE TRANSDUCED THROUGH RELATED PATHWAYS TO DIFFERENTIALLY MODULATE FLT1 ALTERNATIVE SPLICING.

4A: MANUSCRIPT TO BE SUBMITTED FOR PUBLICATION

(References listed at the end of Chapter 4A)

VEGF and FGF2 Signals are Transduced Through Related Pathways

to Differentially Modulate Flt1 Alternative Splicing.

Laura Payne, William R. Huckle*

Department of Biomedical Sciences and Pathobiology Virginia-Maryland College of Veterinary Medicine Virginia Tech, Blacksburg, VA 24061, USA

*Correspondence: [email protected]

ABSTRACT

Alternatively spliced gene products are often associated with human disease. Expression of secreted, inhibitory isoforms of the VEGF-A receptor Flt1, encoded by alternatively-spliced mRNAs, is dysregulated in preeclampsia - a pregnancy-related and potentially fatal disorder marked by hypertension and proteinuria. Levels of placental angiogenic agents VEGF and FGF2 also are altered in preeclampsia, where remodeling of uterine arteries by soluble Flt1-producing embryonic trophoblasts is insufficient to support normal vascular development of the placenta. Here, we report differential impact and of VEGF and FGF2 on Flt1 splice variant transcripts in human umbilical vein endothelial cells. We find that VEGF induces increased relative expression of overall Flt1 mRNA in a concentration-dependent manner, specifically in the forms Flt1 and sFlt1_v1. In contrast, with little impact on overall Flt1 relative expression levels, FGF2 induces a shift in Flt1 splicing toward sFlt1_v2, independent of FGF2 concentration. Through inhibition of key signaling kinases, PI3K, Akt, PKC and MEK, we report that the related VEGF and FGF2 signals are transduced via ERK, using distinct upstream mechanisms. Specifically, our data indicate that VEGF signals through PKC-MEK to activate ERK and, in turn, influence Flt1 splice variant transcripts. Alternatively, the FGF2 signal cascades via PKC-independent mechanisms to activate MEK-ERK. These findings indicate that VEGF and FGF2 signal via related but distinct mechanisms to differentially impact relative expression levels of Flt1, sFlt1_v1 and sFlt1_v2 transcripts.

INTRODUCTION

Changes in eukaryotic gene expression, in response to developmental or environmental cues, may occur via transcriptional, post-transcriptional, or epigenetic mechanisms. Through the occurrence of alternative pre-mRNA processing, the human proteome can be exponentially expanded from roughly 20,000 protein-coding genes [105]. By using exon inclusion/exclusion, alternative 5’ or 3’ splice sites and cleavage/polyadenylation sites, or retention of intronic sequences, alternative processing can generate multiple polypeptides from a single gene.

Transcripts of 95% of mammalian gene are estimated to undergo alternative processing [106-

108]. Since alternative splice patterns are often correlated to specificity and severity of disease, the profiles of splice products have been suggested as viable diagnostic biomarkers [114].

Further, a number of proposed mechanisms aimed at modulating various aspects of alternative splicing are being examined as potential drug targets [115-120, 180]. Although research has directly linked extrinsic stimuli with altered cellular function via changes in splice variant selection for a number of genes [181-184], characterization of the signaling mechanisms that underlie such changes is limited (reviewed [104, 182, 184]). Here, we report growth factor- activated signaling mechanisms leading to alternative splicing of Flt1, a receptor for vascular endothelial growth factor A (VEGF).

VEGF is a potent pro-angiogenic mitogen that influences development, maintenance and remodeling of the vasculature (for reviews on VEGF and its receptors, see [42, 185-190]). VEGF signals through two tyrosine kinase receptors, Flt1 (VEGFR1) and KDR (VEGFR2). Flt1 binds

VEGF with 10-20-fold greater affinity than does KDR [47] but has limited capacity to transduce

VEGF signals independent of KDR [48]. Consequently, Flt1 is thought to act principally as a decoy or sink for VEGF, reducing its availability to KDR. Alternative processing of Flt1 pre-

35 mRNA transcripts yields 3 predominant mRNA isoforms, encoding: 1) a full-length transmembrane protein kinase receptor; 2) sFlt1_v1 – a truncated, secreted/soluble isoform [51]; and 3) sFlt1_v2 - a recently described soluble variant [4, 5, 52] (Figure 1). The sFlt1 isoforms retain only the extracellular Ig-like domains and retain full VEGF binding capacity [47, 56]. The soluble forms of Flt1 perform an additional function as spatial regulators of emerging vessel sprouts [57, 58] via their ability to bind to heparan sulfate in the extracellular matrix [59].

sFlt1 has been associated with preeclampsia (PE) [33, 34, 38, 66, 68], a pregnancy- related and potentially fatal disorder marked by maternal hypertension and proteinuria. Although the etiology of PE remains unclear, remodeling of uterine arteries by sFlt1-producing embryonic trophoblasts is insufficient to support normal vascular development of the placenta [25].

Elevated levels of sFlt1 are found in maternal plasma in PE [33, 37, 191, 192], suggestive of a mechanism involving inhibition of VEGF-driven neovascularization in PE [35]. Consistent with this notion, reduction of circulating levels of sFlt1 alleviates PE-like symptoms in mice [36].

Moreover, anti-VEGF therapies are associated with PE-like symptoms [60, 61], further supporting the postulated role of sFlt1 in PE. However, these studies are reflective of sFlt1_v1 only. Since 2007, reports of a second variant emerged, sFlt1_v2 - a variant not captured in a portion of the sFlt1 measurements of the previous studies. More recent studies indicate that, rather than dysregulated levels of sFlt1_v1, it is the ratio of sFlt1_v1 to sFlt1_v2 that is dysregulated in preeclampsia [4-6, 52].

Factors responsible for dysregulated Flt1 variant expression in preeclampsia are unknown. However, recent studies indicate VEGF is responsible for sFlt upregulation [67, 68].

Here, we report the impact of VEGF on the full-length form of Flt1 (Flt1) and the soluble variants, sFlt1_sv1 and sFlt1_sv2 in human umbilical vein endothelial cells. Additionally, we

36 report impact of fibroblast growth factor 2 (FGF2), another key pro-angiogenic molecule, on Flt1 alternative splicing. We demonstrate that VEGF and FGF2 differentially affect Flt1 splice variant mRNA relative expression levels via related but distinct signaling mechanisms.

EXPERIMENTAL PROCEDURES

Materials. HUVECs (C2519A) and EGM2 (EGM2 BulletKit, CC-3162) were purchased from Lonza.. PacBlue Annexin V conjugate was from Life Technologies (catalog no A35122).

Propidium Iodide was from Thermo Fisher (P3566). DMSO was from Sigma (D8418). Inhibitors used were Wortmannin (Sigma, W1628), MK2206 (Selleckshem, S1078), Bisindolylmaleimide I

(GF 109203X) (Santa Cruz, sc-24003), U0126 (Life Technologies, PHZ1283).Reagents used were rhVEGF-A (Life Technologies, PHC9394), rhFGF2 (Lonza, CC-4113A), rOrf virus VEGF-

E (Cell Sciences CRV007A), rhPlGF (R&D, 264-PG), DPBS (Cellgro, 21-031)

Cell culture. HUVECs were purchased in two separate lots, each pooled from 3 donors.

Cells were grown and maintained in T75 flasks at 37C at 5% CO2, in EGM2. Experiments were conducted from passages 3 to 6, during which expression of Flt variants was stable as measured by qPCR ..Cells were seeded into 60mm dishes at 5000 cells/cm2 36 hours prior to experimental treatment. To produce a state of growth factor withdrawal, cells were washed with DPBS at ten hours prior to planned stimulation, and medium was replaced with “partial” EGM2 (pEGM2), which lacked both FGF2 and VEGF but retained the IGF-1 and EGF growth factor supplements of EGM2. FGF2 and VEGF deprivation were not associated with significant loss of cell viability or induction of apoptosis, as assessed by flow cytometry of Propidium Iodide and Annexin-V stained cells. To initiate growth factor challenge, pEGM2 containing FGF2, VEGF, or both was used to replace cell medium at 0 h. For kinase inhibitor studies, cells were pre-treated for one

37 hour with pEGM2 containing inhibitor or vehicle (0.1% DMSO). Each treatment was run in duplicate plates and biologically replicated 2 -3 times. At 48h, medium was removed and cells lysed with 300uL RNA lysis buffer (Zymo, R1060). Lysates were stored at -20C.

RNA isolation, cDNA preparation and RT-PCR. Total RNA isolated with Quick-

RNA Miniprep kit (Zymo, R1055), according to manufacturer’s instructions. Random oligonucleotide-primed cDNA was synthesized using High-Capacity cDNA Reverse

Transcription Kit (Applied Biosystems, 4368814) using 1ug RNA. cDNA targets, Total Flt1 and

Flt1 (full-length), were detected using Applied Biosytems Gene Expression Assay Mixes (Total

Flt1 - Hs01052961; full-length Flt1 -Hs1052944). Splice variant sFlt1_v1 was targeted with a custom probe design 5’ - FAM-ACAATCAGAGGTGAGCACT-MGBNFQ – 3’ (Applied

Biosystems) and primers F – 5’-TGCCTGCAGAGCCAGGA-3’, R – 5’-

GTGGTACAATCATTCCTTGTGCTTT-3’. Splice variant sFlt1_v2 was targeted with a custom probe design 5’ - VIC-AAGAGCCTGAACTGTATACA-MGBNFQ – 3’ (Applied Biosystems) and primers F – 5’-CGAGCCTCAGATCACTTGGTTT-3’, R – 5’-

GATGACGATGGTGACGTTGATG-3’, used with Taqman Gene Expression Master Mix

(Applied Biosystems, 4370074). HPRT was used as a housekeeping gene (Applied Biosystems,

4326321E). (MGB – minor groove binder; NFG – nonfluorescent quencher) Samples were run in triplicate for Taqman qPCR analysis, using StepOne Plus Real-Time PCR System (Applied

Biosystems).

Statistical analysis. Samples were compared using Mixed-Model Anova with p<0.05 considered statistically significant, p<0.01 considered very significant, and p<0.001 considered extremely significant. In graphs, data are presented as the mean +/- standard error of the mean.

RESULTS

rhVEGFA and rhFGF2 differentially modulate relative mRNA expression of Flt1 mRNA splice variants. Relative expression of Flt1 splice variant mRNAs was examined via qPCR in cells temporarily deprived of FGF2 and VEGF, crucial regulators of endothelial function. VEGF re-stimulation, at concentrations reflective of normal physiology (2ng/mL), did not elicit significant changes in Flt1 variant mRNA relative expression when compared to untreated cells at 48h (Figure 2A, VEGFA). In order to rule out the possibility that VEGF was being sequestered by soluble Flt1 variants in the conditioned culture medium, we treated cells with VEGF-E or placental growth factor (PlGF), representing two different approaches to circumnavigate the possible neutralization of VEGF-A by soluble Flt1 proteins. VEGF-E is a virally encoded VEGF isoform that is selective for KDR, whereas PlGF is selective for Flt1 and, through competitive binding, will render VEGF available to KDR. Neither treatment with

VEGF-E nor PlGF+VEGF-A resulted in significant Flt1 variant mRNA relative expression changes when compared to untreated cells at 48h (Figure 2A).

We reasoned that higher concentrations of VEGF, representing pathological conditions of exposure, may be required to elicit a change in the Flt1 variant signaling pattern. VEGF altered

Flt1 pre-mRNA alternative splicing in a concentration-dependent manner (Figure 2B).

Increasing concentrations of VEGF resulted in corresponding increases in total Flt1 mRNA relative expression. Specifically, total Flt-1 increased 1.45 +/- 0.16 fold when treated with

50ng/ml VEGF, compared to no treatment at 48h. The increase in total Flt1 was comprised of variants Flt1 (1.45 +/- 0.10 fold) and sFlt_v1 (1.61 +/- 0.01 fold). There was no significant change in sFlt1_v2 mRNA relative expression, even at the highest concentration of VEGF.

We next examined the effect of FGF2 on Flt1 pre-mRNA alternative splicing (Figure

2C). In contrast to the lack of expression changes observed in Flt1 variants at low levels of

VEGF, the concentration of FGF2 (4ng/mL) corresponding to that in EGM2 induced a significant change in the Flt1 splice pattern, specifically in increased sFlt1_v2 (1.39 +/- 0.09 fold) compared to no treatment at 48h. Flt1 and sFlt1_v1 were slightly reduced by -0.18 +/- 0.04 fold and -0.12 +/- 0.02 fold, respectively. Although total Flt1 levels increased with higher FGF2 concentrations, total Flt1 relative expression did not change significantly at 100ng/mL FGF2

(0.17 +/- 0.04 fold) compared to no treatment at 48h. However, at 100ng/mL FGF2, the variant splice pattern shifted further toward sFlt1_v2 (1.73 +/- 0.12 fold) with no significant changes in

Flt1 and sFlt1_v1 relative expression compared to no treatment at 48h.

Next, we examined the impact of FGF2 and VEGF in combination. At low levels of

VEGF (2ng/mL) and FGF2 (4ng/mL), the Flt1 variant splice pattern was indistinguishable from the pattern with FGF2 alone, showing a 1.25 +/- 0.06 fold increase in sFlt1_v2, slight decreases in sFlt_v1 (-0.10 +/- 0.04 fold) and Flt1 (-0.20 +/- 0.07 fold), and no change in total Flt1 relative expression. However, when high concentrations of VEGF (50ng/mL) and FGF2 (100ng/mL) were combined, the Flt1 splice variant pattern reflected the VEGF-induced pattern with increased sFlt1_v1 (1.36 +/- 0.19 fold), Flt1 (1.36 +/- 0.15 fold), and total Flt1 (1.37 +/- 0.03 fold), with no change in sFlt1_v2 (Figure 3)

Together, these data indicate that VEGF and FGF2 impact Flt1 pre-mRNA alternative splicing differentially. The VEGF-related increase in relative mRNA expression was restricted to the full-length Flt1 and sFlt1_v1 forms, with no change in sFlt1_v2 levels, whereas FGF2 stimulation shifted the Flt1 splice variant pattern toward sFlt1_v2 with little impact on total Flt1

40 or sFlt1_v1 relative expression levels. The FGF2-induced Flt1 variant pattern predominated in the presence of low levels of VEGF, but was overcome with increasing VEGF concentrations.

VEGF and FGF2 impact Flt1 pre-mRNA alternative splicing via related distinct signal transduction cascades. We next explored signaling mechanisms underlying the differential impact of FGF2- and VEGF on Flt1 pre-mRNA splicing. Specifically, we examined

2 key pathways shared by FGF2 and VEGF - PI3K/Akt and Raf1/MEK/ERK - which are involved in survival and proliferation and may be activated by either PKC- or GRB2-mediated signaling. We probed FGF2 and VEGF activation of Akt and ERK related pathways by pharmacological inhibition of key intermediary kinases. HUVECs were pre-treated with inhibitors targeting PI3K (Wortmannin), Akt (MK2206), PKC (BIM X) and MEK1/2 (U0126), followed by stimulation with either VEGF or FGF2 for 48h. The characteristic VEGF-induced changes in Flt1 splice variant pattern was abrogated by PKC or MEK1/2 inhibitors, whereas neither PI3K nor Akt inhibitors significantly reduced the VEGF-related signal (Figure 4A).

Further, treating cells with phorbol-12-myristate-13-acetate (PMA), an activator of PKC, resulted in a Flt1 variant pattern that resembled an amplified version of the VEGF-related pattern, with increases in relative expression levels of sFlt1_v1 (4.04 +/- 0.17 fold), full-length

Flt1 (3.52 +/- 0.11) and total Flt (4.00 +/- 0.33) and no significant change in sFlt1_v2 compared to control treatments at 48h (Figure 4C).

In contrast to the pattern of inhibitor effects noted under VEGF stimulation, the FGF2- induced Flt1 variant signaling pattern was altered by the Akt inhibitor and abrogated by the

MEK inhibitor, whereas inhibitors of PI3K and PKC did not attenuate the FGF2-induced signal

(Figure 4B). Specifically, MEK, but not PKC inhibition completely abrogated the FGF2-induced shift toward sFlt1_sV2, suggesting that FGF2 activates ERK not via PKC but rather utilizes the

41 classical Grb2/SOS/Ras pathway. Additionally, by blocking PKC-mediated ERK activation, it follows that an increase in the GRB2-mediated ERK activated signal might be seen - supported here with an increase in sFlt1_v2 relative expression of 1.85 +/- 0.12 fold compared to control treatments at 48h (an increase of 1.54 fold from the FGF2-induced signal). Additionally, inhibition of Akt (but not PI3K) altered the FGF2-induced Flt1 variant signaling pattern, although in a manner differing from that of MEK inhibition. Rather than abrogating sFlt1_sv2 mRNA expression, which remained unchanged, inhibition of Akt following FGF2-stimulation resulted in upregulation of total Flt1, specifically in the forms Flt1 and sFlt1_v1 (reflective of the

VEGF/PKC-mediated induction), resulting in a new (s)Flt1 variant pattern. We noted that

VEGF-stimulated Akt inhibition yielded similar increases in total Flt1, also in the forms Flt1 and sFlt1_v1, although these changes were not significant. Consequently, in FGF2- stimulated

HUVECs, Akt inhibition results in magnification of the PKC-mediated Flt1 variant signaling pattern.

FIGURES

Figure 1. Flt1 variants. Flt1 pre-mRNA is alternatively spliced to yield a full-length transmembrane-spanning variant, Flt1, with 7 extracellular immunoglobulin-like domains. Two well characterized soluble variants, sFlt1_v1 and sFlt1_v2, are spliced prior to the transmembrane domain and consist of the extracellular domain. Consequently, they retain full VEGF-A binding capacity. In addition to these 3 Flt1 variants, Real-Time qPCR targets include “total Flt1”, which captures all three variants to measure collective relative expression levels. Exons are represented by numbered boxes. Taqman qPCR primers and MGB probes are indicated by arrows and rectangles, respectively.

A. 1.8

1.6

1.4

1.2 RQ Flt1_sV1 1.0 Flt1_sV2 Flt1 0.8

0.6 VEGFA VEGFE PlGF+VEGFa

Treatments

B. 1.8

** * 1.6 **

* 1.4

sFlt1_V1 1.2 RQ sFtl1_V2 1.0 Flt1 Total Flt1 0.8

0.6 2 10 25 50 rhVEGF-A (ng)

C. 2.0 ** **

1.8 **

1.6 * 1.4 sFlt1_v1

RQ * 1.2 sFlt1_sv2 Flt1 1.0 Total Flt1 0.8 * * 0.6 4 20 50 100 rhFGF (ng/mL)

Figure 2. VEGF-A alters Flt1 pre-mRNA splicing in a dose dependent manner while FGF2 shifts splicing toward sFlt1_v2. HUVECs were deprived of rhVEGF-A and rhFGF2 for 10 hours prior to treatments and were collected at 48h. Relative expression of mRNAs were measured via qPCR, normalized to HPRT and compared to untreated cells at 48h. A) rhVEGF does not elicit changes in Flt1 splice variant levels. HUVECs were treated with rhVEGF-A (2ng/mL), rhVEGF-E (2ng/mL), or rhPlGF (10ng/mL) and rhVEGF-A (2ng/mL) together. No (s)Flt1 splicing response was observed. B.) rhVEGF-A dose response curve. With increasing dosage, rhVEGF-A induces an overall increase in total Flt1 mRNA relative expression, specifically in the Flt1 and Flt1_sV1 forms. C.) rhFGF2 dose response curve. rhFGF2 shifts the Flt1 pre-mRNA splice pattern toward Flt1_sV2, with little increase in total Flt1 mRNA relative expression. Error bars shown as mean +/- SEM; n=2-3 independent experiments; each biological replicate was performed as duplicate treatments. p<0.05*, p<0.01**, p<0.001***

A. 2.0

1.8

1.6

1.4 sFlt1_v1

RQ sFlt1_v2 1.2 Flt1 1.0 total Flt 0.8

0.6 100 ng FGF 50 ng VEGF 100 ng FGF + 50 ng VEGF

B. 1.6

1.4

1.2 sFlt1_v1

RQ sFlt1_v2 1.0 Flt1

0.8 total Flt

0.6 4ng/mL FGF2 2ng/mL VEGF 4ng/mL FGF2 + 2ng/mL VEGF

Figure 3. Combination treatments of rhVEGF-A and FGF2. HUVECs were deprived of rhVEGF-A and rhFGF2 for 10 hours prior to treatments and were collected at 48h. Relative expression of mRNAs were measured via qPCR, normalized to HPRT and compared to untreated cells at 48h. A.) Cells were treated with high levels of either FGF2 (100ng/mL) or rhVEGF-A (50ng/mL) alone or together for 48h. When rhFGF2 and rhVEGFA were combined, the (s)Flt1 splice pattern resembles that of rhVEGF alone. B.) Cells were treated with low levels of either FGF2 (4ng/mL) or rhVEGF-A (2ng/mL ) alone or together for 48h. When rhFGF2 and rhVEGFA were combined, the (s)Flt1 splice pattern resembles that of rhFGF2 alone. Data shown as mean +/- SEM; n=2-3 independent experiments; each biological replicate was performed as duplicate treatments

A. 2.4

2.2

2.0

1.8 sFlt1_V1

1.6 sFlt1_V2 RQ 1.4 Flt1 1.2 TotalFlt 1.0

0.8

0.6 rhVEGF + Wortmannin + MK2207 + GFX + U0126

B. *** ** 2.0

1.8 ** *** 1.6 *** sFlt1_V1 1.4

sFlt1_V2 RQ 1.2 Flt1 TotalFlt 1.0

0.8

0.6 rhFGF2 + Wortmannin + MK2208 + GFX + U0126

Figure 4. Inhibitor studies of Akt and ERK related pathways. rhVEGF and rhFGF2 impact (s)Flt1 relative mRNA expression through related signaling mechanisms. HUVECs were deprived of rhVEGF-A and rhFGF2 for 9 hours prior to addition of Wortmannin (200nM), MK2206 (7.5uM), Bisindolylmaleimide X HCl (BIM X; 2.25uM), U0126 (10uM) or controls (0.1% DMSO) for 60min, followed by treatments with rhVEGF (50ng/mL) or rhFGF2 (4ng/mL) and collection at 48h. Relative expression of mRNAs were measured via qPCR, normalized to HPRT and compared to either DMSO (for rhFGF2 or rhVEGF) or inhibitor-only treatments at 48h. A) rhVEGF-induced relative mRNA expression of (s)Flt1 variants was abrogated by BIM X (PKC) and U0126 (MEK1/2) inhibitors. B) rhFGF2-induced relative mRNA expression of (s)Flt1 variants was abrogated by U0126 (MEK1/2 inhibitor) and altered by MK2206 (Akt inhibitor). Data shown as mean +/- SEM;

n=3 independent experiments; each biological replicate was performed as duplicate treatments *** (p-value <0.001), ** (p-value <-0.01), *(p-value <0.05)

DISCUSSION

VEGF and FGF2 are pro-angiogenic molecules important in the vascular development of the placenta [25, 193-195]. Both VEGF and FGF2 are expressed in placental cytotrophoblasts and syncytiotrophoblasts [17, 196, 197], which are also responsible for dysregulated soluble Flt1 production in preeclampsia [4, 5]. VEGF and FGF2 signal through VEGFR-1 & -2 (Flt1 and

KDR) and FGF Receptors 1-4, respectively, which are members of the tyrosine kinase family of receptors (RTKs), the largest class of cell surface receptors. RTKs receive and transduce extracellular signals (e.g. growth factors) through a related set of signaling networks and, in turn, affect fundamental cellular processes such as cell growth, survival and proliferation [198]. One of the fundamental ways extracellular signals lead to change in cell function is by modifying gene expression, including alternative splicing. Here, we report that FGF2 and VEGF differentially influence Flt1 pre-mRNA spicing. We demonstrate that VEGF-induced increases in total Flt1, Flt1, and sFlt1_v1 mRNA relative expression occurs through PKC/MEK/ERK in a dose dependent manner. Alternatively, FGF2 signals via non-PKC-mediated ERK activation to shift the Flt1 variant splice pattern toward sFlt1_v2 dominance at low and high FGF2 concentrations. Further, Akt may play a role in negative regulation of PKC-mediated impact on

Flt1 variant expression.

We explored the effect of varied concentrations of VEGF and FGF2 on Flt1 alternative splicing. We observed no significant changes in Flt1 variant mRNA levels at concentrations of

VEGF that are sufficient to maintain endothelial cell viability. We explored the possibility that

VEGF was being sequestered from sFlt1s present in the conditioned medium by examining the effects of VEGF-E and PlGF+VEGF on Flt1 variant expression. Neither treatment elicited changes in Flt1 variant expression. These studies confirmed that VEGF does not impact Flt1 splicing at low levels in HUVECs. However, increased concentrations of VEGF did alter Flt1 splicing with corresponding increases in mRNA relative expression of total Flt1 in the forms Flt1 and sFlt1_v1. At the highest concentration, VEGF induced increases of 45%, 45% and 61% in total Flt1, Flt1 and sFlt1_v1 mRNAs, respectively. In contrast, low levels of FGF2 stimulation elicited a 39% increase in sFlt1_v2 relative expression with little impact on total Flt1, Flt1, and sFlt1_v1 – a pattern opposite that observed with VEGF. Although sFlt1_v2 increased by up to

73% the highest FGF2 concentrations, the impact on total Flt1, Flt1 and sFlt_v2 remained insignificant. Moreover, in experiments combining VEGF and FGF2 at low concentrations, the

FGF2-related Ftl1 variant pattern was dominant. This pattern was reversed at high concentrations of VEGF and FGF2, where the VEGF-related Flt1 variant pattern appeared to predominate. We believe this is due the finding that FGF2 does not significantly affect total Flt1 expression levels, but rather shifts the continually-produced Flt1 transcript pool toward sFlt_v2; since high concentrations of VEGF do increase total Flt1 expression, the sFlt_v2 variant becomes a small fraction of the total Flt1 pool. These results indicate that, not only do FGF2 and VEGF induce opposing Flt1 variant patterns, their relative influence is concentration dependent, with FGF2 driving a sFlt1_v2 dominant signal at low concentrations and VEGF driving the Flt1 and sFlt_v1 dominant signal at high concentrations. We reason that, as levels of VEGF rise in preeclampsia, correlated here with an increased sFlt1_v2:sFtl1_v1 ratio, VEGF may drive the sFlt1_v2 upregulation associated with this pregnancy complication. However, it is important to note that Flt1 variant expression is cell-specific, with sFlt1_v2 predominating in non-endothelial

49 cells [5, 67]. Given the findings here in HUVECs, examination of the differential impact of

FGF2 and VEGF on Flt1 alternative splicing in additional cell types, including cytotrophoblasts, would be informative for disease-specific application.

Following our findings that FGF2 and VEGF differentially affect Flt1 pre-mRNA splicing, we thought it possible that FGF2 and VEGFA utilize different signaling mechanisms to impact the Flt1 pre-mRNA target. We examined signaling pathways involving the kinases Akt and ERK, both of which affect myriad downstream substrates leading to altered expression of genes that are involved in proliferation and survival. While both FGF2 and VEGF are reported to activate Akt via the classical PI3K/Akt pathway, they can activate ERK via different upstream effectors. The two mechanisms by which RTKs are known to activate ERK are - 1) a classical

Grb2/SOS-mediated Ras activation, and 2) a Ras-independent mechanism via PLCγ/PKC, both of which lead to Raf1/MEK/ERK cascade. While VEGF activates ERK via the latter mechanism, FGF2 has been shown to utilize either upstream mechanism [71, 84, 199-201]. We explored these pathways by using inhibitors that target PI3K, Akt, PKC and MEK (the only known ERK1/2 kinase [82]). Inhibition of PKC and MEK, but not PI3K or Akt, significantly abrogated the VEGF-induced Flt1 splice pattern, indicating that VEGF impacts Flt1 alternative splicing via the PKC/MEK/ERK cascade. This is consistent with recent findings by Saito et al.

(2013), showing VEGF-stimulated upregulation of sFlt1_v1 via PKC/MEK in human aortic endothelial cells [67]. Alternatively, only inhibition of MEK abrogated the FGF2-induced Flt1 splice pattern, indicating that FGF2 influences Flt1 alternative splicing via MEK/ERK via the non-PKC-mediated pathway, GRB2/SOS/RAS. Together, these data indicate that VEGF and

FGF2 impact Flt1 pre-mRNA alternative splicing via related but distinct signaling cascades leading to activation of ERK. Different upstream initiators of ERK cascade through Raf1,

MEK1/2, ERK1/2, where multiple isoforms and distinct components involved at each level lead to varied activation of the many ERK1/2 substrates [81, 82, 87, 88] and, in turn, varied gene expression responses. Thus, it follows that differential activation of ERK1/2 by VEGF and FGF2 leads to different Flt1 alternative splicing outcomes.

We also noted that FGF2-stimulated Akt inhibition (but not PI3K) resulted in increased mRNA levels of total Flt1, Flt1, and sFlt1_v1 - a pattern reminiscent of the PKC-mediated signal. This suggests regulatory crosstalk between and Akt and the PKC-mediated cascade. In fact, multiple points of crosstalk between these pathways have been described (reviewed [202]).

More specifically, Akt has been demonstrated to regulate Raf1 [203], a component in both classical and non-classical ERK signaling, in a Raf1 isoform-specific manner [204]. Further understanding of the presently implicated role of Akt could provide additional insight into the

(dys)regulation of Flt1 variant production.

In summary, we provide evidence that FGF2 and VEGF differentially influence Flt1 alternative splicing to yield opposing Flt1 splice variant patterns and have differing influences on total Flt1 relative expression levels. FGF2 and VEGF induce their respective changes in Flt1 splicing by signaling through different upstream mechanisms that lead to ERK1/2 activation in

HUVECs. As dysregulation of the soluble forms of Flt1 has recently been characterized in preeclampsia, a deeper understanding of the mechanisms driving Flt1 alternative splicing may eventually lead to therapeutic interventions where none currently exist.

ACKNOWLEGMENTS

The authors thank Dr. Stephen Were for his provision of statistical analysis.

Portions of this study have been presented in poster form at the Annual Vascular Biology

Conference of the North American Vascular Biology Organization, Hyannis, MA on Oct 19,

2015

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4B: SUPPLEMENTARY RESULTS

4B.1 Results 4B.1.1 Physiologically relevant levels of FGF2, but not VEGF, alter Flt1 variant splicing Prior to VEGF and FGF2 dose response experiments (reported in Chapter 4.A, Figure 4.2) we examined the impact of basal levels of VEGF and FGF2 on Flt1 variant mRNA relative expression (Figure 4.5). We observed that the splicing pattern following VEGF treatment was very similar to that of the untreated control at both 24h and 48h. However, FGF2 treatment induced a distinct pattern from the 24h and 48h untreated controls that was not significantly altered when FGF2 and VEGF treatments were combined. Specifically, at 24h the untreated and VEGF-treated samples resulted in relative mRNA expression increases from 0h of sFlt1_v1 (1.57 ± 0.05 fold and 1.54 ± 0.08 fold, respectively) and for Flt1 (1.64 ± 0.09 fold and 1.49 ± 0.06 fold, respectively), with no significant changes in sFlt1_v2. The 24h relative expression of Flt1 variant mRNA in response to FGF2 and FGF2+VEGF were, for sFlt1_v1, 1.34 ± 0.08 fold and 1.40 ± 0.03 fold, respectively; for Flt1, 1.16 ± 0.07 fold and 1.23 ± 0.08 fold, respectively; and for sFlt1_v2, 1.24 ± 0.1 fold and 1.23 ± 0.09 fold, respectively, compared to 0h. Consistent with this, at 48h (compared to 0h), the relative expression levels for Flt1 mRNA were, for untreated, VEGF-treated, FGF2-treated, and combined FGF2+VEGF treatments, as follows: increases in sFlt1_v1 of 1.72 ± 0.09 fold, 1.62 ± 0.07 fold, 1.26 ± 0.11 fold and 1.29 ± 0.07 fold, respectively; increases in Flt1 of 1.90 ± 0.05 fold, 1.96 ± 0.07 fold, 1.26 ± 0.08 fold and 1.30 ± 0.12 fold, respectively; decreases in sFlt1_v2 of 0.47 ± 0.02 fold, 0.44 ± 0.06 fold, 0.79 ± 0.05 fold and 0.84 ± 0.07 fold, respectively. In essence, we did not observe a Flt1 variant splicing response to VEGF-stimulation. In order to investigate this further, we examined the effects of VEGF-E and PlGF + VEGF-A, as detailed in Chapter 4.A (Figure 4.2) where we explained that VEGF does not affect Flt1 variant mRNA relative expression at 2ng/mL in HUVECs. Supplementary to this, we examined the effect of VEGF-E and PlGF on the FGF2-induced signal to see if any of these treatments might alter the Flt1 splice variant pattern observed with FGF2 stimulation (Figure 4.6). Again, there were no significant associated changes in Flt1 variant mRNA relative expression, lending further confirmation that 2ng/mL of VEGF does not influence Flt1 variant mRNA relative expression in HUVECs. Dose response curves to FGF2 and VEGF (Figure 4.2)

57 did reveal that, at higher levels, VEGF treatment results in increased total Flt1 mRNA in the forms Flt1 and sFlt1_v2, while FGF2 stimulation shifts Flt1 splice variant patter toward sFlt1_v2 at both low and high FGF2 concentrations, as detailed in Chapter 4.A.

*** 2.00 *** *** *** *** *** *** *** 1.50 *** *** ** ** ** **

1.00 sV1 RQ sV2 0.50 Flt *** ***

0.00

- - +VEGF +FGF +VEGF+FGF - +VEGF +FGF +VEGF+FGF

0 h 24 h 48h

Sample, Time (hrs)

Figure 4.5. FGF2, but not VEGF, induced Flt1 mRNA variant splice responses. HUVECs were starved of FGF2 and VEGF for 10h prior to treatment with FGF2 (4ng/ml) or VEGF (2ng/mL) for 24h or 48h. Treatment with VEGF did not affect Flt1 splicing when compared to the untreated control at 24h or 48h. Combination VEGF and FGF2 treatments did not affect Flt1 splicing compared the FGF2-treated sample at 24h or 48h. Significant changes in sFlt1_v2 were only observed at 48h. Therefore, we chose this time point for analysis. Each biological sample was run in duplicate. Error bars are represented as ± SEM; n=3. p < 0.05 *, p < 0.01 **, p < 0.001***.

2.5

2.0

1.5

RQ 1.0 sv1 sv2 0.5 flt

0.0

48h,0 VEGFA PlGF VEGFE PlGF+VEGFa PlGF+VEGFe FGF FGF+PlGF FGF+VEGFa FGF+VEGFe F+P+VEGFa

Treatments, 48h

Figure 4.6. Treatments with VEGF-E or PlGF did not uncover a VEGF-A signal or alter the FGF2- induced Flt1 splice variant pattern. HUVECs were starved of FGF2 and VEGF for 10h prior to treatment with FGF2 (4ng/ml), VEGF (2ng/mL), PlGF (10ng/mL), or indicated combinations thereof, for 48h. Neither treatment with VEGF-E nor PlGF + VEGF-A significantly altered the Flt1 splice variant patterns associated with the 48h untreated or FGF2 control treatments. Each biological sample was run in duplicate. Error bars are represented as ± SEM; n=3

4B.1.2 Cell Viability – Inhibitor treatments Following the inhibitor studies outlined in of Chapter 4.A (Figure 4.4), we examined the effect of inhibitors on cell viability and apoptosis (Figure 4.8). (Raw flow cytometry data can be found in Appendix A.) HUVECs were incubated with either Wortmannin (PI3K inhibitor), MK2206 (AKT inhibitor), GFX (PKC inhibitor), U0126 (MEK1/2 inhibitor), SRPIN (SRPK inhibitor), TG003 (Clk inhibitor) or DMSO control, for 60min prior to addition of VEGF or FGF2 for 48h. Cells were stained with Annexin V and PI and subsequently analyzed via flow cytometry as described in Chapter 3. Control cells, treated with DMSO, DMSO + VEGF, or DMSO + FGF2, were 84.3%, 86.1% and 87.4% viable, respectively. Corresponding dead cell percentages were 11.1, 8.4 and 8.0, respectively. Corresponding early apoptotic cell percentages were 1.15, 1.38 and 1.67, respectively. This pattern was consistent across all inhibitor treatments, with the exception of MK2206, with no significant changes observed. Of note, only live cells are reported for GFX (PKC) and U0126 (MEK) inhibitions since both inhibitors fluoresced in the same channels as the chosen dyes - interfering with accurate apoptotic and dead cell counts. MK2206 treated controls were each less than 0.5% viable, with 14.1% of MK2206 treated cells in early apoptosis and 85.8% dead. They were partially rescued by VEGF treatment with 52.7%

59 in early apoptosis and 46.2% dead, and less so by FGF2 with 25.8% in early apoptosis and 71.1% dead. It is important to note, however, when cells in such fragile condition are subjected to the process of harvest, washing and staining for flow cytometry analysis, some portion of cells will not survive. Visual assessment of MK2206-treated cells at 48h confirms a high degree of cell death but does not confirm 0% viable cells. As conditioned medium is removed from adherent cells prior to harvest for RNA analysis, floating dead cells are not included in that analysis as they are here. Therefore, our findings on the effect of Akt inhibition on Flt1 variant mRNA relative expression are derived from live, adherent cells and, we feel, are reliable findings.

100

60 Live Cells

50 Early Apoptotic 40 % Cells % Cells 30 Dead Cells 20

pEGM2+FGF2 TG003 +VEGF pEGM2(+DMSO) pEGM2+VEGF Wortmannin Wortmannin +VEGF Wortmanning+ FGF2 MK2206 MK2206+ VEGF MK2206+ FGF2 GFX GFXVEGF + GFXFGF2 + U0126 U0126+ VEGF U0126+ FGF2 SRPIN SRPINVEGF + SRPINFGF2 + TG003 TG003+ FGF2

Figure 4.8. Effects of inhibition on HUVEC viability. With the exception of MK2206, inhibitor treatments were non-toxic to cells. HUVECS were starved of FGF2 and VEGF at -10h. At -1h, pEGM2 was replaced by pEGM2 + respective inhibitors (Wortmannin (200nM), MK2206 (7.5uM), GFX (2.25uM), U0126 (10uM), SRPIN (50uM), TG003 (10uM) or DMSO (0.1%) controls) for 60m. At 0h, cells were treated with FGF2 (4ng/mL) or VEGF (50ng/ml), harvested at 48h, stained with Annexin V and PI, and analyzed via flow cytometry.

4B.2 Discussion Here, we report supplementary findings to those reported in Chapter 4A. We examined the effect of basal levels of VEGF and FGF2 on Flt1 variant mRNA relative expression at 24h and 48h. FGF2-stimulation resulted in distinct Flt1 splice variant patterns, at both 24h and 48h, whereas treatment with VEGF did not induce any observable changes in Flt1 variant mRNA expression, compared to untreated samples. When VEGF and FGF2 treatments were combined, the Flt1 splice variant signature mimicked that of FGF2 only, at both 24h and 48h. To explore this further, we conducted experiments using VEGF-E treatments and PlGF + VEGFA treatments, as discussed in Chapter 4.A, which did not uncover a VEGF-related signal on Flt1 variant mRNA relative expression. Here, we report that VEGF-E and PlGF + VEGFA treatments, in combination with FGF2, did not alter the FGF2-induced Flt1 splice variant pattern. This is further indication that basal levels of VEGF do not affect Flt1 splicing in HUVECs. In addition, we examined the effects of the inhibitors Wortmannin (PI3K), MK2206 (Akt), GFX (PKC), U0126 (MEK1/2), SRPIN (SRPK) and TG002 (Clk) on HUVEC viability as it corresponds to our experimental layout for mRNA analysis. We found that these inhibitors had no effect on HUVEC viability at 48h, with the exception of MK2206. Although MK2206 was toxic, this was not surprising, given the key role of AKT in cell survival. Of note, our mRNA analysis was performed on adherent cells only, and as VEGF and FGF2 treatments in MK2206- inhibited cells yielded Flt1 splice variant patterns distinct from that of MK2206 alone, we believe the mRNA levels following Akt inhibition findings are meaningful. In summary, we report findings here that are supplementary to those reported in Chapter 4.A. We offer additional data to support the findings that basal levels of VEGF do not affect Flt1 alternative splicing in HUVECs. In addition, we demonstrate that the selected inhibitors used to investigate the signaling mechanisms behind VEGF- and FGF2-induced Flt1 alternative splicing are non-toxic with the exception of MK2206.

4B.3 Methods - Described in Chapter 3 and Chapter 4A.

CHAPTER 5: IDENTIFICATION OF SR PROTEIN CANDIDATES

5.1 Abstract The vast majority of mammalian protein-coding genes each yield multiple proteins through the mechanism of alternative splicing. While many factors influence the process of alternative splicing, SR proteins are known to play a key role. SR proteins are ribonucleobinding proteins with a unique stretch of serine/arginine-rich dipeptides at the C-terminus. Phosphorylation of this region leads to translocation of SR proteins from the cytoplasm to the nucleus, where they bind to pre-mRNA and interact with other splice regulating proteins. In this manner, SR proteins relay upstream signals to effect changes in mRNA splice variant expression. The most well characterized SR protein kinases are the families of Akt, SPRK and Clk (cdc2-like kinase). We have examined SR proteins in HUVECs for a potential role in Flt1 alternative splicing, which becomes dysregulated in preeclampsia - a potentially lethal pregnancy-related disorder. Through bioinformatic prediction tools, we identified two SR protein candidates, SRSF2 and SRSF3, that may be involved in Flt1alternative splicing. RNA analysis indicates that transcript levels of SRSF3 are significantly reduced by FGF2 via MEK activation of ERK. In addition, we examined potential involvement of SR protein kinases through pharmacological inhibition of Akt, SRPK and Clk. Our results suggest that FGF2-induced splicing of Flt1 may cascade through Akt and SRPK.

5.2 Introduction Environmental stimuli can lead to significant alterations in cellular function by inducing changes in splice patterns [181-184]. One of the most prominent signaling mechanisms used to alter splicing of a target gene is by influencing the associated splice-regulatory protein via transcription [184], protein stability/degradation [152, 184, 205-211], localization and accessibility [152, 184, 209, 212-214], altered protein-protein interaction via differential phosphorylation [215, 216], and changes in recruitment and kinetic coupling [216-218]. One major family of splice-regulatory proteins known to play a key role in alternative splicing is the SR protein family. Although changes in transcript levels may contribute to their function, they are largely regulated by phosphorylation state, which dictates SR protein localization and protein interaction [138, 153-157]. The predominant SR protein kinases are Clks, SRPKs, and Akt.

Binding of SR proteins to ESEs can lead to selection of weak splice sites [142]. In general, SR protein binding within an exon promotes exon inclusion while binding to flanking introns encourages exon skipping [144, 145], a pattern that has also been demonstrated with alternative exons [146, 147]. We have applied the models of SR protein binding to bioinformatic-predicted mapping of SR protein binding sites within Flt1 pre-mRNA. Through examination of the resulting predicted binding patterns, we identified potential SR protein candidates that may play a role in Flt1 mRNA alternative splicing. RT-qPCR examination of VEGF- and FGF2-induced SRSF2 and SRSF3 transcripts indicated FGF2-induced regulation of SRSF3 mRNA via ERK signaling. Additionally, we used inhibitor studies to examine FGF2- and VEGF-signaling through the major SR protein kinases, Akt, Clk and SRPK, in Flt1 alternative splicing. Our data indicate that FGF2-signaling cascades through Akt and SRPK to alter Flt1 splicing.

5.3 Results 5.3.1 Classical SR Proteins are present in HUVECs First, we examined HUVECs for presence of SR protein mRNAs. We designed endpoint primers (Table 5.4) for the classical SR proteins that, when possible, distinguished between reported splice variants. Additionally, we designed targeted primers (Table 5.4) for the SR protein kinases, Akt1-3 isoforms, Clk1-3 isoforms, and SRPK1-2 isoforms. We detected all 7 classical SR proteins and both SRPK1 and 2. Of the 3 Clk isoforms, we detected Clk2 and 3. Of the 3 Akt isoforms, we detected Akt1 and 3 (Figure 5.1). The specific coding variants captured via endpoint PCR are listed in Table 5.3. The inability to detect Clk1 and Akt2 may be due to either non-expression in HUVECs or ineffective primers. However, given that Akt2 is typically found in highly insulin-sensitive tissues, it may be possible that Akt2 is not significantly expressed in HUVECs. Tissue specificity for Clks is not well documented.

A.) B.)

C.) D.)

E.) F.)

Figure 5.1 Detection of SR proteins in HUVECs. cDNA from HUVECs maintained in EGM1 were examined via Endpoint PCR for presence of the classical SR proteins, Akt1/2/3, Clk1/2/3, and SPRK1/2. A) Detections of SRSF1, SRSF3 and SRPK1; B) detection of SRSF2 and SRSF7; C) Detection of SRSF4, SRSF5 and SRSF6; D) Detection of Akt1, Akt3 but not Akt 2; E.) Detection of SRPK2 and Clk3; F)

Detection of Clk2 but not Clk1; Positive control (Flt1). All bands corresponded to expected sizes.

5.3.2 Inhibition of SR protein kinases on Flt1 alternative splicing. We examined the role of Akt, Clk and SRPK in Flt1 alternative splicing via pharmacological inhibition (Figure 5.2). Cells were incubated with MK2206 (Akt1/2/3 inhibitor), SRPIN (SRPK1/2 inhibitor), or TG003 (Clk 1/2/4) prior to treatment with VEGF, FGF2 or DMSO controls for 48h. Akt inhibition (MK2206) of FGF2-stimulated cells, as discussed in detail in Chapter 4.A, results in highly significant increases in mRNA expression levels of total Flt1 in the forms Flt1 and sFlt1_v1 mRNAs at 48h, compared to FGF2-treated cells. Here, we show that inhibition of SRPK and Clk does not significantly abrogate the VEGF- induced Flt1 splice variant pattern. However, inhibition of SRPK does abrogate the FGF2- induced splice variant pattern when compared to controls at 48h. Specifically, total Flt1 increases 1.10 ± 0.03 fold compared to SRPK only - a highly significant increase from the FGF2-induced reduction of 0.82 +/- 0.03 fold. Flt1 and sFlt1_v1 mRNA expression levels of 1.06 ± 0.04 fold and 1.04 ± 0.04 fold are both highly significant increases from the FGF2-induced signals of 0.76 +/- 0.05 fold and 0.87 +/- 0.02 fold, respectively. We did observe a decrease in sFlt1_v2 levels,

65 although it was not-significant at 1.16 ± 0.06 fold compared to the FGF2-only signal of 1.31 +/- 0.02 fold. In addition, Clk inhibition following FGF2 induction resulted in sFlt1_v2 relative expression that is significantly changed from the FGF2 increase if 1.31 +/- 0.2 fold, to an increase of 1.67 ± 0.04 fold compared to controls at 48h. This is a 1.36-fold increase from that of the FGF2-only sample. These data suggest that the FGF2-stimulated Flt1 splice variant pattern may be induced through AKT and/or SRPK phosphorylation of SR proteins, while Clk phosphorylation of SR proteins may function to downregulate the FGF2-induced signal – a supported concept of SRPK/Clk functional differences [219]. Akt, SRPK and Clk have been demonstrated to work in concert or independently [9, 10, 100, 163-166, 219]. In summary, the VEGF-induced Flt1 splice variant pattern is not significantly affected by Akt, SRPK, or Clk inhibition and therefore does not appear to impact Flt1 pre-mRNA alternative splicing via direct SR protein phosphorylation. In contrast, the FGF2-induced Flt1 splice variant pattern is highly significantly altered by Akt and SRPK inhibition. Further, sFlt1_v2 is significantly magnified by Clk inhibition These findings suggest a direct role for phosphorylated SR proteins in FGF2- induced Flt1 pre-mRNA splicing via Akt, SRPK and Clk regulation, which, in the case of the latter, appear to play differential roles. Further studies to elucidate the specific roles of Akt, SRPK and Clk in mediating FGF2-induced Flt1 splicing via SR proteins would inform the concerted mechanisms by which Akt, SRPK, and Clk collectively alter Flt1 splice variant expression.

2.4

2.2

2.0

1.8

1.6 sFlt1_V1

1.4 sFlt1_V2 RQ 1.2 Flt1 TotalFlt 1.0

0.8

0.6

rhVEGF +MK2207 +SRPIN +TG003

2.0 B.) * *** ** 1.8

1.6 ** ** *** ** 1.4

sFlt1_V1

RQ sFlt1_V2 1.2 Flt1

1.0 TotalFlt

0.8

0.6

+TG003 rhFGF +MK2208 +SRPIN

Figure 5.2. Effects of SR protein kinase inhibition on Flt1 alternative splicing. HUVECs were starved of FGF2 and VEGF at -10h. Cells were exposed to MK2206 (7.5 uM), SRPIN (50uM), TG003 (10uM), or DMSO (0.1%) for 60m prior to treatment with rhVEGF (50ng/mL) or rhFGF2 (4ng/mL) for 48h (samples were performed in duplicate). Relative expression of mRNAs were measured via RT-qPCR, normalized to HPRT and compared to either DMSO or inhibitor-only treatments at 48h. A.) VEGF treatments. Inhibition of Akt (MK2206), SRPK1/2 (SPRIN) or Clks (TG003) did not abrogate the VEGF-induced Flt1 splice variant pattern. B.) FGF2 treatments. Inhibition of Akt (MK2206) and SRPK1/2 (SPRIN) altered the FGF2- induced Flt1 mRNA variant expression levels of sFlt1_v1, Flt1 and total Flt1. Clk (TG003) inhibition resulted in amplification of the FGF2-induced increase in sFlt1_v2 mRNA relative expression. Data shown as mean +/- SEM; n=3. *** (p-value <0.001), ** (p-value <-0.01), *(p-value <0.05)

5.3.3 Akt, but not ERK, is predicted to phosphorylate SR proteins Next, we examined the 20 known SR proteins (listed in Table 5.1) for predicted Akt and ERK phosphorylation motifs. For this we used the prediction site designed by J. Obenauer, L. Cantely and M Yaffe at Massachusettes Institute of Technology [220], Scansite 3, which searches for motifs within proteins that are likely to be phosphorylated by specific kinases or to bind specific domains. The phosphorylation sites are predicted using “the matrix of selectivity values for amino acids at each position relative to the phosphorylation site as determined from the oriented peptide library technique described by Songyang et al 1995” [221]. We searched the amino acids sequences of each SR protein listed in Table 5.1 using Akt and Erk Scansite 3 motifs represented in Figure 5.3. Our searches resulted in no predicted matches for Erk kinase phosphorylation, indicating indirect regulation of SR proteins or use of other splice regulatory mechanisms to affect pre-mRNA splicing. Of the 20 SR proteins examined for predicted Akt phosphorylation motifs, 19 returned at least one site prediction (Figure 5.4). The number of predicted Akt phosphorylation sites for each SR protein is listed in Table 5.1.

Table 5.1 List of known SR proteins examined for predicted Akt and ERK phosphorylation motifs. The 20 known SR proteins were examined for presence of predicted Akt and ERK phosphorylation motifs. Of these, 19 are predicted to be phosphorylated by Akt and none are predicted to be phosphorylated by ERK. Listed are aliases for each SR protein, the Uniprot identifier number, the number of sites which are predicted to be phosphorylated by Akt and, in the final column, whether the SR protein is found in the SpliceAid 2 database (see section 5.3.5) and if it was included in species alignment (see section 5.3.4).

Gene/Protien UniProt # of predicted Searchable in Aliases Name Accession No Akt p-motifs SplicAid2/Aligned SRSF1 SF2/ASF, SRp30a Q07955 6 y/y SRSF2 SC35. SRp30b, SFRS2A Q01130 13 y/y SRSF3 SRp20 P84103 5 y/y SRSF4 SRp75, SFRS4 Q08170 7 y/n SRSF5 SRp40, SFRS5, HRS Q13243 11 y/y SRSF6 SRp55. SFRS6 Q13247 6 y/y SRSF7 9G8, AAG3 Q16629 11 y/y SRSF8 SRp46, SFRS2B Q9BRL6 2 n/n SRSF9 SRp30c, SFRS9 Q13242 1 y/y SRSF10 FUSIP, TASR, SRp38, SFRS13 O75494 5 y/y SRSF11 P54, SFRS11, NET2 Q05519 4 y/y SRSF12 SRrp35, SFRS19, SFRS13B Q8WXF0 6 n/n hTRA2α AWMS1 Q13595 7 y/y hTRA2β SFRS10 P62995 4 y/y RNPS1 LDC2 Q15287 4 n/n SREK1 SFRS12, SRrp508, SRrp86 Q8WXA9 4 n/n U2AF35 U2AFBP, FP793, U2AF1 Q01081 0 n/n U2AF65 U2AF2 P26368 1 n/n SnRNP70 U170K, Snp1, RU17 P08621 1 n/n PRKA17A XE7, SFRS17A, AK17A Q02040 4 n/n

A.) B.)

Figure 5.3 Scansite Motif logos for Akt and Erk . Motif logos are graphical representation of an aligned set of sequences, in which the frequency of an amino acid at each position is represented by the height of the letter and the sequence conservation is represented by the total height of the stack. These are motif logos used by Scantsite 3 to score the likelihood of an input sequence to be phosphorylated by A) Akt or B) ERK.

A.) B.)

C.) D.)

E.) F.)

G.) H.)

I.) J.)

K.) L.)

M.) N.)

O.) P.)

Q.) R.)

S.)

Figure 5.4. Predicted Akt phosphorylation sites in SR proteins. Using Scansite 3 [220], we searched all 20 known SR proteins for the presence of predicted Akt phosphorylation motifs. Results are presented as graphs and depict amino acid locations for high stringency predicted phosphorylation sites, as well as mapped domains such as RRM (RNA recognition motif). Predicted surface accessibility is also represented, as calculated from the sequence and is not referenced to known structures. The protein name is depicted under each graph (A-S) and predicted phosphorylation sites are detailed in Table 5.2

5.3.4 Sites of predicted Akt phosphorylation in SR proteins are largely conserved In the interest of investigating which of the predicted Akt phosphorylation sites are most likely to be valid, we examined the predicted sites of in 12 SR proteins (based on those we were able map to Flt1 pre-mRNA due to availability in SpliceAid 2, see section 5.3.5 and Table 5.1) for species conservation. Using Uniprot Clustal Omega for multiple species alignments (http://www.uniprot.org/align/), we aligned human (Homo sapiens), chimp (Pan troglodytes), mouse (Mus musculus), chicken (Gallus gallus), western clawed frog (Xenopus tropicalis) and zebrafish (Danio rerio) sequences. When information for chimp, chicken, western clawed frog or zebrafish could not be found, we used orangutan (Pongo abelii), common turkey (Meleagris gallopavo), African clawed frog (Xenopus laevis) or Japanese whitefish (Oryzias latipes), respectively. Table 5.2 details information on each predicted phosphorylated amino acid location for each of the 12 SR proteins, including the specific amino acid residue, number of examined species in which it is conserved, species in which it is unaligned, as well as the amino acid substitution (conserved substitutions are included). Raw alignments can be found in Appendix C. Of the 74 predicted Akt phosphorylation sites aligned, there were a total of 11 unconserved

74 substitutions in 10 sites, as well as 11 potentially skipped sites, predominantly in zebrafish and frog. With these exceptions, the predicted Akt phosphorylation sites in the SR proteins we examined are highly conserved.

Table 5.2. Conservation of predicted Akt phosphorylation sites in SR proteins. For the indicated SR proteins, alignments were performed for human, chimpanzee or orangutan, mouse, chicken or common turkey, western clawed frog or African clawed frog, and zebrafish or Japenese whitefish. Listed are: the predicted Akt phosphorylation amino acid residue being aligned; how many species of the 6 examined are in alignment; which species is not conserved; and, if there is a substitution, which amino acid is substituted.

Predicted Aligned in Unaligned Gene name AKT phos Substitution all? species aa residue SRSF1

s211 y

s213 5 of 6 chicken A

s215 Y

s217 Y

s219 Y

s221 Y

SRSF2

s128 Y

s130 Y

s132 Y

s134 Y

s136 Y

s138 Y

s140 Y

s159 Y

s161 Y

s185 Y

s187 Y

s189 5 of 6 frog T

frog - T; fish s191 4 of 6 frog, fish - skipped SRSF3

s120 5 of 6 frog skipped

s122 Y

s128 5 of 6 frog L

Predicted Aligned in Unaligned Gene name AKT phos Substitution all? species aa residue s130 5 of 6 frog K

s138 5 of 6 frog Y

SRSF5

s192 Y

chicken, t194 3 of 6 frog, S

zebrafish s196 Y

s198 Y

s204 Y

mouse, s219 4 of 6 skipped chicken s225 Y

s229 Y

s263 5 of 6 chicken skipped

s265 Y

s267 5 of 6 frog skipped

SRSF6

s206 Y

s218 Y

s220 Y

s230 5 of 6 frog T

s332 5 of 6 mouse skipped

s342 Y

SRSF7

s130 Y

s132 Y

s134 Y

s144 5 of 6 fish G

s167 5 of 6 fish A

s175 4 of 6 frog, fish T, skipped

s183 5 of 6 fish

s200 Y

s202 Y

s204 4 of 6 chick, fish V, A

s225 Y

SRSF9

s199 Y

SRSF10

Predicted Aligned in Unaligned Gene name AKT phos Substitution all? species aa residue s123 5 of 6 fish A

s131 5 of 6 fish skipped

s133 5 of 6 fish A

s160 5 of 6 fish D

s253 5 of 6 fish skipped

SRSF11

s262 Y

s278 Y

s308 5 of 6 fish skipped

s350 Y

TRA2A

mouse; s75 4 of 6 skipped fish s84 Y

s86 Y

T88 5 of 6 fish S

s98 Y

s274 Y

s276 Y

TRA2B

s85 Y s87 Y s280 Y s282 Y

5.3.5 Predicted SR protein binding to Flt1 pre-mRNA We examined the reported Flt1 genomic sequence (Accession no: AADB02015943), including coding sequences for exons 13, 14, 15a, 16 and flanking intronic sequences, for predicted SR protein binding motifs. For this we used SpliceAid 2 [222], a database of human splicing factors expression data and RNA target motifs. SpliceAid 2 searches exact motifs versus a database of strictly experimentally assessed target RNA sequences. The submitted sequences are processed and exact correspondence with the database sequences is identified. Of the 19 SR proteins that Akt is predicted to phosphorylate, 12 were available for examination in SpliceAid 2, (see Table 5.1 and Table 5.3), including all the classical SR proteins. With the exception of SRSF4, all the examined SR proteins were predicted to bind to Flt1 pre-mRNA. We mapped this information to a schematic of relevant Flt1 pre-mRNA sequences. Using the general guideline -

77 binding within exons leads to exon inclusion and binding to intronic sequences which flank exons leads to exon skipping - we assessed potentially influential patterns for each of the 11 SR proteins predicted to bind to Flt1 pre-mRNA (Figure 5.5). Using these methods, we summarized SR protein mapping to Flt1 pre-mRNA in Table 5.3. Based on unpatterned predicted binding patterns, SRSF4-11 and hTRA2A/B were eliminated as potential Flt1 pre-mRNA alternative splicing candidates. SRSF1 showed predicted binding with the 3’ unique CDS (2x) and 3’UTR (24x) in sFlt1_v1 and also within sFlt1_v2 flanking introns (1x on each side) of exon 15a and in 3’UTR (3x). This pattern indicates inclusion of sFlt1_v1 and sFlt1_v2 exclusion. SRSF2 showed predicted binding in sFlt1_v1 exon13 (1x), unique CDS (2x) and 3’UTR (33x) and also flanking sFlt1_v2 unique exon 15a (3x in i14 and 4x in i15a) and in 3’UTR (6x). As with SRSF1, this pattern indicates sFlt1_v1 inclusion and sFlt1_v2 exclusion. In addition, SRSF2 was of particular interest because 5 of the predicted binding sites with sFlt1_v1 pre-mRNA were predicted by ScanSite 2 to be silencing rather than enhancing. As this is not frequently seen with SR proteins, coupled with the key locations of SRSF2 predicted binding, we selected this as one of our top candidates for further investigation. SRSF3 was predicted to bind in each section of sFlt1_v1; however, it also returned 45 predicted binding sites within sFlt1_v2 unique exon 15a and 67 in the 3’ UTR. Of note, no other SR proteins were predicted to bind to exon 15a. Consequently, we selected SRSF3 as a top candidate with SRSF2. (Complete SpliceAid 2 results, including the predicted binding sequence/location, experimental references, and PubMed ID, can be found for each SR protein in Appendix D.)

A.)

B.)

C.)

D.)

E.)

F.)

G.)

H.)

I.)

J.)

K.)

Figure 5.5. SR protein maps to Flt1 pre-mRNA. Using SpliceAid 2 [222], Flt1 genomic sequence was examined for predicted binding by SR proteins. A) Overview map of all SR proteins examined. B) SRSF1, C) SRSF2 – ultimately chosen as a top candidate due to predicted binding in sFlt1_v1 Exon13 and 3’UTR, while flanking sFlt1_v2 Exon 15a. D) SRSF3 – ultimately chosen as a top candidate due to very high predicted binding with sFlt1_v2 Exon 15a and 3’UTR. Additionally, SRSF3 was predicted to function both 82

as a silencer (in red) and enhancer. E) SRSF5, F) SRSF6, G) SRSF7, H) SRSF9, I) SRSF10, J) SRSF11, K) hTRA2A/B

Table 5.3 Summary of SR protein predicted binding in portions of sFlt1_v1 and sFlt1_v2 pre-mRNA sequences. Predicted binding was assessed using SpliceAid2 [222]. Columns for sFlt1_v1 (sv1) and sFlt1_v2 (sv2) sequences are divided into blue coding exons (Ex[#]), blue unique sv1 3’ coding sequence (3’CDS), blue 3’ untranslated regions (3’UTR), and green flanking intronic sequences (i[#]). Numbers in columns represent the number of binding sites predicted for each SR protein. SR proteins in red represent those with patterns of interest and those in bold red were ultimately chosen as top candidates. The “*” indicates an SR protein which was predicted to have both enhancer and silencer activity. PB = predicted binding.

SR sFlt1_v1 sFlt1_v2 Summary Protein Ex13 3’CDS 3’UTR i13 i14 Ex15a 3’UTR i15a PB in sv1 3’CDS/UTR, SRSF1 2 24 1 3 1 flanks sv2 PB in sv1 SRSF2* 1 2 33 3 6 4 exon/3’CDS/UTR, flanks sv2 SRSF3 1 3 40 1 45 67 2 Very high PB in sv2 Ubiquitous PB - SRSF5 4 3 40 3 1 6 eliminated Low PB in sv1 UTR - SRSF6 2 eliminated Low PB in sv1 UTR - SRSG7 3 eliminated Ubiquitous PB - SRSF9 4 11 59 3 3 13 3 eliminated Low PB in sv1 UTR/intron and in sv2 SRSF10 11 1 3 1 exon/UTR/intron- eliminated SRSF11 2 2 Low PB in sv1 UTR and * intron - eliminated PB in sv1 hTRA2 1 11 2 1 1 exon/UTR/intron and in A sv2 intron/exon - eliminated PB in v1 exon/UTR/intron and in hTRA2B 1 18 2 2 3 sv2 intron/exon - eliminated

5.3.6 VEGF- and FGF2-signaling and SRSF2 and SRSF3 mRNA levels We examined the effect of FGF2 and VEGF stimulation on the relative expression of SRSF2 and SRSF3 mRNAs (Figure 5.6). HUVECs were starved of VEGF and FGF2 for 10 prior to treatment with VEGF or FGF2 doses for 48h.VEGF stimulation, from 2ng/ml to 50ng/mL had no significant effect on SRSF2 with increases of 1.07 ± 0.07 fold and 1.02 ± 0.03

83 fold respectively. Similarly, there was no significant VEGF-induced SRSF3 response at any dose, with a slight decrease of 0.93 ± 0.02 fold at 50ng/mL at 48h compared to untreated controls. FGF2-stimulation of 4ng/mL did not significantly impact SRSF2, with a decrease of 0.94 ± 0.01 fold. However, FGF-stimulation generally drives down SRSF3 levels, with significant reduction of 0.78 ± 0.02 fold at 20ng/mL compared to untreated controls at 48h. These data indicate that VEGF does not significantly alter SRSF2 or SRSF3 mRNA relative expression at 48h in HUVECs, while SRSF3 mRNA relative expression is significantly reduced by FGF2-stimulation at 20ng/mL compared to untreated cells. Our mapping of predicted binding to Flt1 pre-mRNA revealed a high number of SRSF3 predicted binding sites within exon 15a, which is unique to sFlt1_v2. Our data also indicates that FGF2-stimulation shifts Flt1 alternative splicing toward sFlt1_v2. Collectively, this suggests one mechanism by which FGF-2 induces sFlt1_v2 mRNA expression is by impacting SRSF3 concentration levels. Analysis of FGF-2 induced SRSF3 concentration changes on the protein level would provide an additional layer of confirmation. Next, we measured relative expression of SRSF2 and SRSF3 following inhibition of the VEGF or FGF2 signaling intermediates (Figure 5.7). We used inhibitors that, in our studies, were associated with altered VEGF- or FGF2-induced Flt1 splicing patterns. HUVECs were placed in pEGM2 at -10h and treated with respective inhibitors at -1h. VEGF or FGF2 were added at 0h and cells harvested at 48h. First we examined VEGF-stimulated inhibition of MK2206 (Akt), GFX (PKC), U0126 (MEK) (Figure 5.7a). VEGF-induced SRSF2 and SRSF3 mRNA relative expressions resulted in decreases of 0.88 ± 0.01 fold and 0.86 ± 0.01 fold, respectively, compared to DMSO controls at 48h. Inhibition of the VEGF-induced signal did not results in any significant changes when compared to VEGF-induced mRNA levels of SRSF2 or SRSF3. Specifically, Akt inhibition of the VEGF signal resulted in a increase from the VEGF- only signal in SRSF2 to 0.94 ± 0.04 fold, compared to the Akt inhibitor control at 48h. Inhibition of MEK following VEGF induction resulted in decreases in both SRSF2 and SRSF3 to 0.67 ± 0.02 fold and 0.67 ± 0.03 fold, compared to the MEK inhibitor control at 48h. Inhibition of PKC resulted in complete abrogation of the VEGF-induced SRSF2 and SRSF3 mRNA relative expression, (0.99 ± 0.07 fold and 0.99 ± 0.02 fold, respectively) compared to the PKC inhibitor control at 48h. Together, these data suggest that the observable, although non-significant, VEGF influence on SRSF2 and SRSF3 mRNA relative expression is mediated by PKC. Next, we

84 examined FGF-stimulated inhibition of MK2206 (Akt), U0126 (MEK) and SRPIN (SRPK). Given the relationship of the MEK/ERK signaling pathway to PCK, we also examined PKC inhibition for FGF2 signaling (Figure 5.7b). FGF2 stimulation, alone, resulted in decreases in SRSF2 and SRSF3 of 0.89 ± 0.01 fold and 0.81 ±0.05 fold, respectively, compared to DMSO controls at 48h. Inhibition of Akt resulted in a non-significant increase in SRSF3 from the FGF2- only signal to 0.93 ± 0.07 fold and unchanged SRSF2, compared to the Akt inhibitor control at 48h. Inhibition of PKC did not significantly alter FGF2-induced SRSF2 or SRSF3 mRNA levels compared to the FGF2-induced signal at 48h. Inhibition of SRPK did not significantly alter SRSF3 mRNA levels but resulted in an extremely significant decrease in SRSF2 to 0.76 ± 0.01 fold, compared to the FGF2-only decrease of 0.89 ± 0.01 fold. Inhibition of MEK resulted in a complete reversal of FGF2-induced SRSF2 and SRSF3 relative mRNA expression, with increases of 1.28 ± 0.1 fold and 1.10 ± 0.09 fold, respectively, compared to the MEK inhibitor control at 48h. The increases in SRSF2 and SRSF3 following MEK inhibition of the FGF2 signal are extremely significant compared to the FGF2-only decreases of 0.89 ± 0.01 fold and 0.81 ± 0.05 fold, respectively, These data suggest that FGF2 signaling impacts SRSF2 and SRSF3 relative expression via MEK. Collectively, these findings suggest that FGF2 significantly reduces SRSF3 mRNA expression and that FGF2 impact on SRSF2 and SRSF3 occurs via a PKC-independent MEK activation mechanism. We did not find that VEGF signaling has a significant influence on SRSF2 and SRSF3 mRNA levels. However, the minor SRSF2 and SRSF3 responses to VEGF were completely abrogated by PKC, but not MEK. In fact, inhibition of VEGF- and FGF2- induced MEK have opposite effects on SRSF2 and SRSF3 levels – reflective of the differential VEGF and FGF2 impact on Flt1 variant splicing via ERK.

B.)

A.) 1.25 1.25

1.00 1

SRSF2 RQ SRSF2

SRSF3 SRSF3 RQ

0.75 0.75 * 2 10 25 50 4 20 50 100 rhVEGFA, (ng/mL) rhFGF (ng/mL)

Figure 5.6. FGF2- and VEGF- induced SRSF2 and SRSF3 mRNA relative expression in HUVECs at 48h. HUVECs were starved from VEGF and FGF2 for 10h prior to treatment with either A) VEGF (2ngmL – 50ng/mL) or B) FGF2 (4ng/mL – 100ng/mL) for 48h. Relative expressions of SRSF2 and SRSF3 mRNAs were assessed following cDNA preparation and amplification via Real-Time qPCR. Samples were referenced to untreated controls at 48h and normalized to HPRT. Each biological sample was run in duplicate. Error bars are represented as ± SEM; n=2. p<0.05*

*** * 1.50 1.50 *

1.25 1.25

1.00 1.00 RQ

SRSF2 RQ SRSF2 SRSF3

0.75 SRSF3 0.75

0.50 0.50

Figure 5.7 Regulation of SR protein mRNA by GFX-mediated VEGF stimulation and ERK- mediated FGF2 stimulation. HUVECs were placed in pEGM2 at -10h, treated with inhibitors at -1h (MK2206 (7.5uM), GFX (2.25uM), U0126 (10uM), SRPIN (50uM) or controls (0.1% DMSO). At 0h, FGF2 (4ng/mL) or VEGF (50ng/mL) was added for 48h. Following RNA and cDNA processing, relative expression was assessed for SRSF2 and SRSF3 mRNAs. Relative expression of mRNAs were measured via qPCR, normalized to HPRT and compared to either DMSO (for rhFGF2 or rhVEGF) or inhibitor-only treatments at 48h. A) VEGF-induced SRSF2 and SRSF3 mRNA relative expression was abrogated by PKC (GFX) inhibition, but not MEK (U0126) or Akt (Mk2206) inhibition. B) FGF2-induced SRSF2 and SRSF3 mRNA relative expression was reversed by U0126 (MEK) inhibition, but not by Akt (MK2206), GFX (PKC) or SRPK (SRPIN) inhibition. . Each biological sample was run in duplicate. Error bars are represented as ± SEM; n=3. p<0.05*, p<0.01**, p<0.001*** 86

5.4 Discussion In efforts to elucidate the mechanisms by which VEGF and FGF2 influence Flt1 pre- mRNA alternative splicing, we were interested in identifying RNA-binding protein candidates that may play a direct role in the related Flt1 alternative splicing events. SR proteins are well known for their role in alternative splicing with a growing number of reports linking them to growth-factor induced splicing [9, 11, 12, 100, 223]. Additionally, we examined the roles of predominant SR protein kinases in Flt1 alternative splicing via inhibition studies. We confirmed the presence of the 7 classical SR protein mRNAs in HUVECs by endpoint PCR. All 7 classical SR proteins were present. We also checked for presence of isoform-specific SR protein kinases AKT 1/2/3, Clk 1/2/3, and SPRK 1/2. We detected them all with the exceptions of Akt2 and Clk1. This may reflect that these isoforms are not present in HUVECs or primer designs were ineffective. However, given the tissue specificity of Akt isoforms and in particular, the expression of Akt2 in highly insulin-sensitive tissues, it is quite possible that Akt2 mRNA is not detectable in HUVECs. In the interest of learning more about direct SR protein regulation in VEGF- or FGF2- induced Flt1 pre-mRNA splicing, we examined the inhibitory effect of SR protein kinases, Akt, SRPK and Clk, on Flt1 variant mRNA relative expression. We found that VEGF-induced Akt, SRPK and Clk inhibition did not affect Flt1 variant mRNA relative expression. However, FGF2- induced Flt1 variant mRNA levels, with the exception of sFlt1_v2, were highly significantly altered by Akt and SRPK inhibition. Further, sFlt1_v2 was magnified by Clk inhibition. These results suggest that VEGF influence on Flt1 alternative splicing does not alter Flt1 pre-mRNA splicing via SPRK-, Akt- or Clk-mediated SR protein phosphorylation. Conversely, FGF2 influence on sFlt1_v1, Flt1, and total Flt1 transcript levels were affected by inhibition of Akt and SRPK, whereas sFlt1_v2 was altered via Clk inhibition – suggesting differential roles for Clk vs Akt and SRPK in related SR protein phosphorylation events. Next, we examined each of the 20 known SR proteins for the presence of predicted Akt and ERK phosphorylation sites using Scansite 3 [220]. Unlike Akt, ERK is not a known kinase of SR proteins, although it has been demonstrated to phosphorylate other RNA-binding proteins [224]. However, our search returned no predictions of direct SR protein phosphorylation by ERK. Alternatively, of the 20 SR proteins searched, 19 contained at least one (and as many as

13) predicted Akt phosphorylation sites. We aligned these sites in 6 species, from human to zebrafish, to find that the predicted Akt phosphorylation sites were highly conserved – an indication they are likely viable phosphorylation sites. Although Scansite 3 results included predicted surface availability, examination of SR protein surface crystal structures would be highly informative, in terms of which of the predicted Akt phosphorylation sites are genuine. We then examined Flt1 pre-mRNA for predicted SR protein binding motifs using SpliceAid 2 [222]. Of the 19 SR proteins with predicted Akt phosphorylation motifs, SpliceAid 2 provided 12 for whose motifs we were able search – the 7 classical SR proteins and SRSF9, SRSF10, SRSF11, hTRA2α and hTRA2β. Searches for predicted binding of each of these resulted in predicted binding sites to Flt1 pre-mRNA for 11 SR proteins, with the exception of SRSF4. Mapping of the results to a schematic representation of Flt1 pre-mRNA to identify emerging patterns that corresponded to exon/intron definition yielded two SR proteins whose predicted binding patterns appeared influential, SRSF2 and SRSF3. The SRSF2 predicted binding pattern was distinct due to its increased frequency within the unique CDS of sFlt1_v1 combined with predicted binding in the introns flanking exon 15a of sFlt1_v2 – a pattern that suggests preference of sFlt1_v1 over sFlt1_v2. The predicted binding pattern of SRSF3 stood out due to its very high predicted binding frequency within exon 15a and 3’UTR, unique to sFlt1_v2. We examined the effect of FGF2 and VEGF on the relative expression of SRSF2 and SRSF3 mRNAs and found that FGF2-stimulation significantly reduced SRSF3 mRNA relative expression. Our inhibition studies indicate that FGF2 regulation of SRSF3 occurs via PKC- independent activation of ERK. Consequently, we found this to be the same mechanism by which FGF2 influences Flt1 variant mRNA splicing. Mapping of SRSF3 to Flt1 pre-mRNA reveals a high number of predicted binding sites within exon 15a and 3’UTR, which is unique to sFlt1_v2. FGF2-stimulation shifts Flt1 splicing toward sFlt1_v2, which suggests that FGF2 signaling may impact Flt1 alternative splicing via regulation of SRSF3 concentrations through PKC-independent ERK signaling. Although expression levels of SR proteins are one way through which they may be regulated, their phosphorylation levels that are considered to affect their localization and function within the cell. Attempts to discern VEGF- or FGF2-stimulated changes in SR protein phosphorylation state have thus far been unsuccessful.

In summary, we identified candidate SR proteins which may be involved in VEGF- or FGF2-induced Flt1 alternative splicing through the use of bioinformatics, inhibitor studies, andqPCR. We identified SRSF2 and SRSF3 as potential candidates via bioinformatics. Collectively, our data indicate that FGF2 induces a shift in Flt1 alternative splicing toward sFlt1_v2 via PKC-independent EKR activation. Moreover, via this same pathway, FGF2 stimulation significantly reduces SRSF3 levels, which is the only SR protein predicted to bind to sFlt1_v2’s unique exon 15a, which it does with a very high predicted frequency. Furthermore, our data suggest that FGF2 impacts sFlt1_v1, Flt1 and total Flt1 mRNA relative expression via Akt- and SRPK-mediated SR protein phosphorylation, while alternatively influencing sFlt1_v2 expression via Clk-mediated SR protein phosphorylation. While our data suggest that VEGF influences Flt1 pre-mRNA splicing via PKC-MEK, we did not find a significant impact of VEGF on SRSF2 or SRSF3 mRNA levels, although we did observe abrogation of observed SRSF2 or SRSF3 mRNA level changes via PKC inhibition. Similarly, we did not find significant evidence for VEGF-induced activation of the SR protein kinases, Akt, SRPK or Clk. Knockdown of these SR proteins would provide more detailed confirmation of the direct role of SR proteins in Flt1 alternative splicing.

5.5 Methods 5.5.1 Materials Inhibtiors SRPIN and TG003 were from Santa Cruz. Beta mercaptoethanol, HEPES, NaF and Na3VO4 were from Sigma. Glycine, HBSS, Methanol, NaCl, Tris, Tween20 were from Fisher Scientific. EDTA was from Boston Bioproducts.

5.5.2 Cell culture, Endpoint PCR and Real-Time qPCR Culturing and PCR Procedures are described in Chapter 3. Primers for endpoint PCR are listed in Table 5.4. Primers and probes for Taqman PCR targeting SRSF2 and SRSF3 were obtained as predesigned Assay Mixes from Applied Biosystems (catalog no 4351372 and no 4331182, respectively). SRSF2 and SRSF3 amplification efficiencies were validated in simplex and duplex qPCR (Figure 5.8).

40 Slopes: SRSF2(sim): -3.40 = 97% SRSF2(dup): -3.46 = 95% 36 SRSF3(sim): - 3.29 = 101% SRSF3(dup): - 3.42 = 96%

Ct 28 SRSF2_sim 24 SRSF3_sim SRSF2_dup 20 0.6 1.6 2.6 3.6 4.6 SRSF3_dup RNA, log equivalents (pg)

Figure 5.8. Efficiencies of simplex vs duplex RT-qPCR for SRSF2 and SRSF3 primer and probe pairs. Amplification efficiencies were compared using a single cDNA target per qPCR well (simplex) or two mRNA targets per well (duplex). SRSF2 and SRSF3 had very similar amplification efficiencies in simplex and duplex qPCR.

Table 5.4. Primers for SR Protein Endpoint PCR. Primer designs for the 7 classical SR proteins and SR protein kinases, Akt1-3, Clk1-3 and SRPK1-2. When possible, primers were designed to distinguish between coding variants for each target, some of which code for unique proteins. If primers were designed to distinguish variants, the captured variant(s) in HUVEC endpoint PCR is listed.

Detected Distinguished Target Forward Primer Reverse Primers in EP Variants PCR SRSF1 5’-GTGAAGCAGGTGATGTATGT-3’ 5’-CGAAGGGAATGTAGATGTTAGG-3’ 2 of 3 (3rd is nc) SRSF2 5’- CGTGTATTGGAGCAGATGTAT-3’ 5’- GAAGTCGTTCACCTCACTAAA-3’ 2 of 3 (3rd is nc) SRSF3 5- AGAGAGTTGGTTGGTGTTG-3’ 5’- GGACGGCTTGTGATTTCT-3’ 2 of 2 SRSF4 5’- GATGCAGATGATGCTGTTTATG-3’ 5’- CTTCGGCTTCTGCTCTTAC-3’ Only 1 form known Y SRSF5 5’ GCTAAGTGCGTCAGTTGT-3’ 5’- GGTGGAGCATTTCGTCTATC-3’ 2 of 2 SRSF6 5’- CTCCTCGAAGTAGACCTCAAA-3’ 5’- CACCTGCTTGTCGCATAAA-3’ 2 of 2 SRSF7 5’- GTCACGGTCTAGATCACATTC-3’ 5’- GAGAGCTTCAGTCCATTCTTT-3’ 2 of 2 Akt1 5’- GAGACTGACACCAGGTATTT-3’ 5’- CCATAGTGAGGTTGCATCT-3’ 3 – not distinguished Y Akt2 5’- CCCAGTCCATCACAATCA-3’ 5’- TGGAAGGAAGCCCTAGTA-3’ 3 – not distinguished N Akt3 5’- ATGTAGATACTCCAGAGGAAAG-3’ 5’- GTAGATAGTCCAAGGCAGAG-3’ 3 – not distinguished Y 2 – not distinguished Clk1 5’- GAAACGTTGTCTGGAATGAG-3’ 5’- GTCTACCTCCCGCTTTATG-3’ N (3&4 are nc) Clk2 5’- CTCACCTACAACCTAGAGAAG-3’ 5’- GAAACTGTGTGGATGGAATAG-3’ Only 1 form known Y Clk3 5’- AGCCAACAGAGCAGTAAG-3’ 5’- GGAGTCTTGGAGCATGTAA-3’ 2 – not distinguished Y SRPK1 5’-GAAGCGAATGCAGGAAATTG-3’ 5’-TCCAGTGGTCCGTTATGT-3’ 1 of 2 (2 is nc) Y SRPK2 5’- CAGGAACTTGCGAACATAGA-3’ 5’- CGGGATCAGGAAATCTGTAAA-3’ 3 – not distinguished Y

5.5.3 Scansite Scansite 3 is located at http://scansite3.mit.edu/#home. We scanned proteins by accession number and looked for the selected motifs Akt Kinase, Erk1 Kinase, as well as Erk D-domain. We used high stringency parameters.

5.5.4 Uniprot Species Alignment Uniprot Clustal Omega multiple species alignment is located at http://www.uniprot.org/align/. We aligned sequences for human (Homo sapiens), chimp (Pan troglodytes), mouse (Mus musculus), chicken (Gallus gallus), frog (Xenopus tropicalis or laevis), and zebrafish (Danio rerio) sequences, or substituted orangutan (Pongo abelii), common turkey (Meleagris gallopavo) or Japanese whitefish (Oryzias latipes) when necessary.

5.5.5 SpliceAid 2 SpliceAid2 is located at http://193.206.120.249/splicing_tissue.html. We entered our sequences of interest and scanned it for predicted binding by each SR protein, one by one. We then manually mapped this onto a schematic representation of the Flt1 pre-mRNA.

5.5.6 Cell culture and treatments for phospho-proteins HUVECs were plated at 10,000cells/cm2 in 10cm plates for 36h in 5% CO2 and 37C. At -10h, adherent cells were washed with DPBS and placed in pEGM2. At -1h, pEGM2 was replaced with H/B/H (HBSS, 25mM HEPES, 0.1% BSA) containing 0.1% DMSO control or respective inhibitors - Wortmannin (200nM), MK2206 (7.5uM), GF 109203X (GFX; 2.25uM), U0126 (10uM), SRPIN (50uM) or TG003 (10uM). At 0h, cells were treated with VEGF (50ng/mL) or FGF2 (4ng/mL) for 90min. Cells were harvested by placing on ice and removal of treatment H/B/H, which was replaced with 1ml RIPA buffer (Pierce, 89900) containing 1mM NaF, 1mM Na3V04, 0.1M EDTA and 1% Protease Inhibitor Cocktail (Sigma, P8340). Lysates were manually scraped, collected in a 1.5mL Eppendorf tube and rotated, end over end, at 4C for 1h. Lysates were centrifuged at 14,000 x g at 4C for 10m. Supernatants were placed in new 1.5mL Eppendorf tubes and stored at -80C.

5.5.7 SDS-PAGE and Immunoblotting Frozen HUVEC lysates were thawed in an ice water bath. Each sample was mixed at a 1:1 ratio with Laemmli Sample Buffer (Bio-Rad, 161-0737) containing 2-mercaptoethanol and boiled for 5m at 96C. Forty microliters of each sample was loaded into wells of Criterion TGX 12% Precast Gels (Bio-Rad, 567-1043). Gels were run at 175V for 1h, followed by 15m shaking at 4C in Transfer Buffer (10% 30.3g Tris, 144.1g glycine; 10% methanol) . Immobilon-FL PVDF membrane (Millipore, IPFL0010) was activated by rocking in methanol for 2m, followed by rocking in transfer buffer for 5m. Proteins were transferred from the gel to the PVDF membrane in a Biorad Criterion Cell at 100V for 1h. The membranes were rinsed in TBS (0.5M Tris-base, 1.55M NaCl, ph7.6) and rocked in Blocking Buffer (Rockland, MB-070) overnight at 4C. The blocked membranes were washed for 5m in TBST (TBS with 0.1% Tween20). Membranes were incubated, rocking, with 1:1000 dilutions of anti-phophoepitope SR protein clone 1H4 (Millipore, MABE50), or Beta Actin (Cell Signaling, D6A8) in 1 part blocking buffer and 1 part TBST for 4 to 6h at 4C, followed by 4 washes in TBST, 5m each. Membranes were then incubated at 1:10,000 with 2o antibodies IR Dye 700 or 800, (Rockland, 611-732-127 and 610-730-124) in 1 part blocking buffer and 1 part TBST, rocking for 1h at room temperature. Membranes were washed 4x in TBST for 5m each, followed by a TBS rinse.

5.5.8 Statistical analysis Samples were compared using Mixed-Model Anova or with Kruskal-Wallis Test using NPAR1WAY procedure, with p<0.05 considered statistically significant, p<0.01 considered very significant, and p<0.001 considered extremely significant. In graphs data are presented at the mean +/- standard error of the mean

CHAPTER 6: DISCUSSION

Alternative splicing (AS) is the process by which exons may be retained or spliced out of pre-mRNA to yield variant mRNAs that, in turn, can lead to multiple proteins from one gene. Alterations in splice patterns are often associated with disease [109-112, 180, 225] and a number of proposed mechanisms aimed at modulating various aspects of alternative splicing (AS) are being examined as potential drug targets [115-120, 180]. AS often occurs in response to extracellular signals. Consequently, elucidation of pathways connecting signal-induced responses to alternatively spliced genes is ongoing. Currently characterizations leading to specific gene targets include, Slo [226-228], NRI [229], CD44 [224, 230-235], CD45 [236-239], FN [100, 104, 240-245], PKC [9] , E1A [166], 4.1R [246], and KLF6 [247]. Here, we describe the effect of FGF2 and VEGF signaling mechanisms on Flt1 alternative splicing. We describe development of a cell culture model and RT-qPCR assay in which alterations in Flt1 splicing could be readily detected. We report differential impact of VEGF and FGF2 on Flt1 splicing via related but distinct signaling mechanisms at various levels, including key kinase intermediates, SR protein kinases and splice-regulating SR proteins. In the preeclamptic placenta, cytotrophoblasts give rise to a syncytial layer that coats the embryonic terminal villi – the site of oxygen and nutrient exchange between mother and fetus. Cytotrophoblasts also adopt an endothelial cell type and invade the maternal spiral arteries in a process crucial for subsequent vascularization of the placenta. In preeclamptic placentas, in which vascular development is insufficient, cytotrophoblasts are responsible for secretion of dysregulated soluble Flt1 variants 1 and 2 [4-6]. We explored the possibility of conducting our studies in primary cytotrophoblasts, but given their very short lifespan in culture, we were unable to do so. We examined two choriocarcinoma lines, BeWo and JEG3, and were unable to obtain adequate levels of Flt1 splice variant mRNAs. We then selected human umbilical vein endothelial cells (HUVECs) which were easy to obtain in pooled donor lots, were relevant in the sense of being embryonic-derived endothelial cells, and expressed the targets of interest adequately. We examined multiple possible growth conditions, including DMEM and EGM1 with modified levels of BBE and FBS. Ultimately, we empirically established an experimental culture environment in EGM2 from which rhFGF2 and rhVEGF were removed 10hours prior to, and for the duration of, 48h treatments. Analysis via flow cytometry showed that the

93 experimental conditions were not cytotoxic. We also assessed the impact of the experimental conditions on cell cycles and found that they were not altered, regardless of VEGF or FGF2 presence. Further studies in an additional cell type would provide necessary information regarding the cell-specificity of FGF2 and VEGF impact on Flt1 alternative splicing responses. Of note, in the course of our preliminary studies we observed a significant impact of FGF2 on Flt1 splice variant expression. Although FGF2 was not included in our original hypothesis, we included it in our remaining experimental studies. We examined the effect of basal levels of VEGF and FGF2 on relative mRNA expression of Flt1 splice variants. We observed that VEGF did not elicit a Flt1 variant splicing response at basal levels, whereas basal FGF2 treatments induced a clear and consistent variant mRNA expression pattern. To explore if the lack of Flt1 splicing response to VEGF could be attributable to sequestration by soluble Flt1 in conditioned medium, we conducted experiments using VEGF- E and PlGF+VEGF. VEGF-E binds only to KDR, not Flt1, circumventing issues of sFlt1 sequestration. Conversely, PlGF binds specifically to Flt1, not KDR, theoretically freeing VEGF for KDR binding. In either case, if the lack of VEGF-induced Flt1 alternative splicing were due to binding by sFlt1 in the culture medium, an existing signal would be uncovered. However, neither treatment with VEGF-E or PlGF+VEGF resulted in Flt1 variant expression changes. We concluded that basal levels of VEGF do not elicit changes in Flt1 splice variant mRNA relative expression in HUVECs. We then examined whether higher concentrations of VEGF or FGF2 influence Flt1 variant mRNA expression. Dose response experiments showed that FGF2 induced a similar response by Flt1 spice variants to both low and high doses – namely, a shift toward sFlt1_v2 with little overall increase in total Flt1 expression. The lack of Flt1 splicing response to basal levels of VEGF was increasingly overcome with higher doses. Converse to the FGF2-induced signal, high concentrations of VEGF induced an overall increase in total Flt1, specifically in the forms Flt1 and sFlt1_v1, when compared to untreated samples. When FGF2 and VEGF were examined in combination, we observed that the FGF2-induced Flt1 splice pattern persists at basal levels and the VEGF-induced Flt1 splice pattern persists at high levels reflective of pathology. In preeclampsia, the sFlt1_v2:sFlt1_v1 ratio increases throughout pregnancy. In the case of HUVECs, our data indicate that FGF2, rather than VEGF, contributes to that shift. Consequently, as VEGF and FGF levels rise, as they do in preeclampsia, the VEGF-induced signal

94 predominates, according to our data. Such a scenario would reflect increases in sFlt1_v1 and Flt1 rather than sFlt1_v2. This highlights one of the limitation of the use of cells that are not derived from trophoblasts. It has been demonstrated that sFlt1_v1 and sFlt1_v2 are expressed in a cell- specific manner, with sFlt1_v2 predominating in non-endothelial cells [5]. Therefore, examination in an additional cell type, preferably trophoblasts, is warranted. Recent studies report VEGF-induced upregulation of soluble Flt1 in murine trophoblast stem cells [68]and human vascular endothelial cells [67]. As these studies examined sFlt1_v1 only, our findings support this. Here, we demonstrate that VEGF affects total Ftl1 relative expression in a variant- specific manner in HUVECs by increasesing mRNA relative expression of sFlt1_v1 (and Flt1) while down regulating sFlt1_v2 expression. Thus, we feel that, to get a clear picture of the contributions of soluble Flt1 to pathologies, both variants must be examined. Given our findings of differential impacts of VEGF and FGF2 on Flt1 pre-mRNA splicing, we examined the related signaling mechanisms via pharmacological inhibition. We targeted key kinases in the PI3K-Akt and PKC-MEK pathways. Our results suggest that VEGF impacts Flt1 pre-mRNA splicing via PKC-MEK activation of ERK. It has been demonstrated that FGF can activate MEK/ERK via PKC-dependent or PKC-independent mechanisms [71, 93]. Our findings indicate that FGF2 activates MEK/ERK via PKC-independent signaling. Together these findings suggest that VEGF and FGF signaling differentially activate the core cascade unit of ERK, Raf1/MEK/ERK, via different upstream mechanisms. Distinct upstream kinases lead to activation of various isoforms within the Raf1/MEK/ERK cascade and also influence adaptor and scaffold proteins, all of which lead to differential activation of the diverse and numerous ERK1/2 substrates. Our data suggest that VEGF and FGF activate ERK1/2 via different upstream mechanisms, leading to differential splicing of Flt1 pre-mRNA. Additionally, inhibition of Akt, following FGF2 stimulation resulted in significantly increased levels of total Flt1 mRNA relative expression, specifically Flt1 and sFlt1_v1. This is reflective of the response observed with PKC-mediated ERK activation, as seen with VEGF or PMA stimulation. It is possible that FGF2-induced Akt may negatively regulate PKC-mediated Flt1 splicing via mechanisms such as crosstalk with the PCK pathway [102, 103] or via SR protein phosphorylation. This opens an interesting avenue for future investigation of FGF2 and VEGF signaling intersections and cross-regulation, as they are often regarded as working in concert with one another [76, 77].

As direct regulators of pre-mRNA splicing, we identified candidate SR proteins which may play a role in VEGF- or FGF2-induced Flt1 alternative splicing. We first confirmed the presence of the classical SR proteins (SRSF1-7), as well as predominant SR protein kinases in HUVECs. Through endpoint PCR, we detected all classical SR proteins in an isoform specific manner, as well as SRPK1/2, Akt 1/3, and Clk 2/3. We then examined all SR proteins in Scansite 2 for predicted phosphorylation by Akt and ERK. Akt has been shown to phosphorylate SR proteins directly [9, 12, 100] and indirectly via Clk [9] and SRPK [165, 166]. As Akt is activated by growth factor signaling, it is not surprising that Akt has been shown to link growth factor signals to alternative splicing via SR proteins [100, 166]. Although ERK has not been demonstrated to phosphorylate SR proteins, it has been shown to activate other RNA-binding splice regulatory proteins [248, 249]. Our searches for predicted EK phosphorylation motifs in SR proteins returned no matches. Akt phosphorylation motifs returned matches for 19 out of 20 SR proteins, (the exception being U2AF35). While some SR proteins, (SRSF9, U2AF65 and SnRNP70) contained only 1 predicted Akt phosphorylation site, others contained as many as 11 (SRSF5 and SRSF7) or 13 (SRSF2). We then examined Flt1 genomic sequences corresponding to, and surrounding, the unique variant exonic sequences for predicted binding motifs of the SR proteins that Akt was predicted to phosphorylate and which were available for searches within the SpliceAid 2 database. Of the 12 SR proteins searched, all except SRSF4 were predicted to bind to Flt1 pre-mRNA. We mapped the binding locations of each of 11 SR proteins to Flt1 pre- mRNA to assess for patterns which may support the general concept of “binding within and exon promotes inclusion and binding in flanking introns promotes skipping”. From this, we identified SRSF2 and SRSF3 as strong candidate SR proteins for involvement in Flt1 alternative splicing. Examination of SRF2 and SRSF3 mRNA relative expression following VEGF and FGF2 dosage stimulation revealed that FGF2-induced significant reduction in SRSF3 mRNA relative expression. Inhibition studies indicate that this occurs through PKC-independent activation of ERK, which is the same mechanism by which FGF2 impacts Flt1 variant splicing, according to our findings. Our mapping data indicates a high number of predicted SRSF3 binding sites within the sFlt1_v2-unique exon 15a, to the exclusion of all other searched SR proteins, and 3’UTR. Given that FGF2-stimulation shifts Flt1 splicing toward sFlt1_v2, this suggests that FGF2 signaling may impact Flt1 alternative splice shift to sFlt1_v2 via regulation of SRSF3 mRNA expression levels via a PKC-independent mechanisms. Probing SR protein phosphorylation

96 states following VEGF and FGF2 stimulation, with or without PI3K/Akt and PKC/MEK inhibition, using a mAb able to detect phosphoepitopes of SRSF1-6 was inconclusive. We also examined the potential involvement of SR protein kinases Akt, SRPK and Clk via pharmacological inhibition. We observed no abrogation of the VEGF-induced Flt1 splicing pattern with inhibition of Akt, SRPK or Clk. As VEGF appears to influence Flt1 splicing via PKC/MEK/ERK, and our findings indicate that ERK does not directly activate SR proteins, this data suggest that VEGF does not directly influence Flt1 pre-mRNA splicing via SR proteins. Conversely, inhibition of SRPK did abrogate the FGF2-induced Flt1 splice pattern via increases in sFlt1_v1, Flt1 and total Flt, indicating that FGF2 may impact these forms through SRPK activation of SR proteins. Akt inhibition altered the FGF2-induced Flt1 splice pattern in a similar fashion, resulting in increased expression sFlt1_v1, Flt1 and total Flt1. Conversely, inhibition of FGF2-induced Clk resulted in amplification of sFlt1_v2. Together, these data suggest that FGF2 impacts Flt1 alternative splicing differentially through SR protein kinases Akt and SRPK vs Clk. Concurrently, our data suggest that FGF2 signaling via PKC-independent ERK activation significantly reduces mRNA relative expression of SRSF3, which is predicted to influence sFlt1_v2 levels.

CHAPTER 7: CONCLUSIONS Alternatively spliced variants are increasingly implicated in various disease states [109- 112, 180, 225]. Consequently, understanding how exogenous cues direct expression of alternative mRNAs and proteins is a pursuit of great importance, potentially leading to targeted molecular therapies at various levels of the tranduced signal [115-120, 180]. Additionally, alternative splice variants are being used as viable diagnostic markers [114]. In particular, soluble Flt1 levels are currently used to predict development of preeclampsia [250]. It is understood that dysregulated soluble variants of Flt1 are involved in preeclampsia; however, the factors that cause this dysregulation are unknown. We present here, findings of signaling mechanisms that Flt1 pre-mRNA splicing. We have developed a suitable cell culture model of HUVECs in modified EGM2, in which to ask fundamental questions regarding growth-factor simulated alternative splicing of Flt1 pre-mRNA. In this model, we are able to quiet expression of Flt1 mRNA variants without serum-starvation and without cytotoxicity, creating a baseline for measurable stimulated expression of Flt1spice variants. From these conditions, we found that VEGF stimulates Flt1 variant mRNA relative expression in a dose dependent manner, increasing the total expression of Flt1 and sFlt1_v1. We also observed that FGF2 influences Flt1 alternative splicing by shifting the splice pattern toward sFlt1_v2, conversely to VEGF-induction. Moreover, we demonstrate that VEGF and FGF2 impact Flt1 pre-mRNA splicing via differential activation of ERK. In addition, our findings indicate that FGF2, but not VEGF, influences Flt1 alternative splicing via regulation of transacting factors on two levels – mRNA levels and post-translational modification. Specifically, our data suggest that FGF2 signaling significantly reduces SRSF3 levels via PKC-independent ERK activation – the same mechanism by which FGF2 encourages inclusion of exon 15a, which is unique to sFlt1_v2. Mapping of SRSF3 predicted binding sites to Flt1 pre-mRNA revealed a high number of predictions in the exon 15a, to the exclusion of all other SR proteins which predicted to be phosphorylated by Akt. Additionally, results from our inhibition studies suggest that FGF2 regulates levels of sFlt1_v1 and Flt1 via Akt and SRPK phosphorylation of SR proteins, while regulating the Flt1 splicing toward sFlt1_v2 via Clk- mediated SR protein phosphorylation. Collectively, these findings suggest that 1) FGF2 signaling shifts Flt1 splicing toward sFlt1_v2 via regulation of SRSF3 transcript levels through

PKC-independent ERK activation and 2) FGF2-induced Flt1 variant splice pattern is altered differentially via SRP protein kinases, Akt, SRPK and Clk. We conclude here that VEGF and FGF2 differentially impact Flt1alternative splicing via related but distinct signaling mechanisms through ERK. Our findings suggest that PKC- independent activation of ERK by FGF2 regulates concentrations SRSF3 which influences the consequent shift in Flt1 alternative splicing toward sFlt1_v2. In additional, FGF2 activates the Akt pathway to differentially influence post-translational modification of SR proteins via SR protein kinases, Akt, SRPK and Clk. We did not find significant influence of VEGF treatments on either the mRNA or post-translational phosphorylation modification levels of SR proteins. Perhaps VEGF signaling operates through constitutive splicing mechanisms to influence Flt1 pre-mRNA splicing, while FGF2 activates alternative splicing mechanisms. Our findings provide new insights into the regulation of Flt1 pre-mRNA splicing, including identification of influential exogenous growth factors, signal transduction pathways and characterization of SR protein regulation at the mRNA and post-translational modification levels. These findings create fertile ground for further investigations into Flt1 variant dysregulation associated with preeclampsia or other relevant vascular pathologies. Future directions for studies should include investigation in a trophoblast-like cell type, knock-down of key kinases and SR proteins, and relationship studies such a UV crosslinking and point mutations. Such investigations, together with these findings, may lead to life-saving therapeutic targets for women and their growing fetuses who suffer from pre-eclampsia, where none currently exist.

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APPENDIX A – AnnexinV/PI Flow Cytometry Raw Data (for Figure 3.6)

sample # EGM2 pEGM2 DMSO Wort MK2206 GFX U0126 SRPIN TG003 VEGF FGF Unstained cells - EGM2 PI stained cells - EGM2 annexin stain - EGM2 1 x 2 x x 3 x 4 x x 5 x 2ng/ml 4ng/ml 6 x x 2ng/ml 4ng/ml 7 x x 50ng/ml 8 x x 4ng/ml 9 x 10 x x 11 x x 12 x 13 x x 14 x x 15 x 16 x x 17 x x 18 x 19 x x 20 x x 21 x 22 x x 23 x x 24 x 25 x x 26 x x

Raw Flow Cytometry Data for Figure 3.6.

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APPENDIX B – Cell Cycle Raw Flow Cytometry Data (for Figure 3.7)

Time VEGF FGF2 (hr) Sample EGM2 pEMG2 (50ng/ml) (4ng/ml) -10h: 1 x 2 x 0h: 3 x 4 x 5 x 6 x x 7 x x 8 x x x 48h: x 9 x (2ng/mL) x 10 x x 11 x x 12 cntrl

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APPENDIX C – Species Alignments for Predicted Akt Phosphorylation Sites in SR Proteins (for Table 5.2)

SRSF1

SP|Q07955|SRSF1_HUMAN MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVE 60 SP|Q5R7H2|SRSF1_PONAB MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVE 60 SP|Q6PDM2|SRSF1_MOUSE MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVE 60 SP|Q5ZML3|SRSF1_CHICK MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVE 60 SP|Q6DII2|SRSF1_XENTR MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVE 60 SP|Q6NYA0|SRS1B_DANRE -MSGGVIRGPAGNNDCRIYVGNLPPDIRTKDVEDVFYKYGAIRDIDLKNRRGGPPFAFVE 59 .****************************:****************************

SP|Q07955|SRSF1_HUMAN GGGGGAPRGRYGPPSRRSENRVVVSGLPPSGSWQDLKDHMREAGDVCYADVYRDGTGVVE 161 SP|Q5R7H2|SRSF1_PONAB GGGGGAPRGRYGPPSRRSENRVVVSGLPPSGSWQDLKDHMREAGDVCYADVYRDGTGVVE 161 SP|Q6PDM2|SRSF1_MOUSE GGGGGAPRGRYGPPSRRSENRVVVSGLPPSGSWQDLKDHMREAGDVCYADVYRDGTGVVE 161 SP|Q5ZML3|SRSF1_CHICK GGGGGAPRGRYGPPSRRSEYRVIVSGLPPSGSWQDLKDHMREAGDVCYADVFRDGTGVVE 161 SP|Q6DII2|SRSF1_XENTR GGGGGAPRGRYGPPSRRSEYRVVVSGLPPSGSWQDLKDHMREAGDVCYADVFRDGTGVVE 180 SP|Q6NYA0|SRS1B_DANRE GGGVGAPRGRYGPPSRRSEYRVIVSGLPPSGSWQDLKDHMREAGDVCYADVFRDGTGVVE 160 *** *************** **:****************************:********

SP|Q07955|SRSF1_HUMAN FVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRSRSRSRSRSNS 221 SP|Q5R7H2|SRSF1_PONAB FVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRSRSRNRSRSNS 221 SP|Q6PDM2|SRSF1_MOUSE FVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRSRSRSRSRSNS 221 SP|Q5ZML3|SRSF1_CHICK FVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSVVVAEAVVGATA 221 SP|Q6DII2|SRSF1_XENTR FVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRSRSRSRSRSNS 240 SP|Q6NYA0|SRS1B_DANRE FVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRS--RSRSRSNS 218 ************************************************ . :.:

SP|Q01130|SRSF2_HUMAN MSYGRPPPDVEGMTSLKVDNLTYRTSPDTLRRVFEKYGRVGDVYIPRDRYTKESRGFAFV 60 SP|Q5R1W5|SRSF2_PANTR MSYGRPPPDVEGMTSLKVDNLTYRTSPDTLRRVFEKYGRVGDVYIPRDRYTKESRGFAFV 60 SP|Q62093|SRSF2_MOUSE MSYGRPPPDVEGMTSLKVDNLTYRTSPDTLRRVFEKYGRVGDVYIPRDRYTKESRGFAFV 60 SP|P30352|SRSF2_CHICK MSYGRPPPDVEGMTSLKVDNLTYRTSPDTLRRVFEKYGRVGDVYIPRDRYTKESRGFAFV 60 TR|Q6P366|Q6P366_XENTR MSYGRPPPDVEGMTSLKVDNLTYRTSPETLRRVFEKYGRVGDVYIPRDRYTKESRGFAFV 60 TR|Q7ZV13|Q7ZV13_DANRE MSYGRPPPDVEGMTSLKVDNLTYRTSPETLRRVFEKYGRVGDVYIPRDRYTKESRGFAFV 60 ***************************:********************************

SP|Q01130|SRSF2_HUMAN RFHDKRDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHSRRGPPPRRYGGGGYGRRSR 120 SP|Q5R1W5|SRSF2_PANTR RFHDKRDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHSRRGPPPRRYGGGGYGRRSR 120 SP|Q62093|SRSF2_MOUSE RFHDKRDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHSRRGPPPRRYGGGGYGRRSR 120 SP|P30352|SRSF2_CHICK RFHDKRDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHSRRGPPPRRYGSSGYGRRSR 120 TR|Q6P366|Q6P366_XENTR RFHDKRDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHGRRGPPPRRYGD--YGRRSR 118 TR|Q7ZV13|Q7ZV13_DANRE RFHDKRDAEDAMDAMDGALLDGRELRVQMARYGRPPDAHYSRRGAPPRRYGGYGRRSRSR 120 ******************:******************:*:.*** ****** ***

SP|Q01130|SRSF2_HUMAN SPRRRRRSRSRSRSR--SRSRSRSRYSRSKSRSRTRSRS--RSTSKSRSARRSKSKSSSV 176 SP|Q5R1W5|SRSF2_PANTR SPRRRRRSRSRSRSR--SRSRSRSRYSRSKSRSRTRSRS--RSTSKSRSARRSKSKSSSV 176 SP|Q62093|SRSF2_MOUSE SPRRRRRSRSRSRSR--SRSRSRSRYSRSKSRSRTRSRS--RSTSKSRSARRSKSKSSSV 176 SP|P30352|SRSF2_CHICK SPRRRRRSRSRSRSR--SRSRSRSRYSRSKSRSRTRSRS--RSTSKSRSARRSKSKSSSV 176 TR|Q6P366|Q6P366_XENTR SPRRRRRSRSRSKSR--SRSRSRSRYSRSKSRSRTRSRT--RSSSKSRSARRSKSKSSSA 174 TR|Q7ZV13|Q7ZV13_DANRE SPRRRKHSRSRSRSRSRSRSRSRSRYSRSRSRSYSRSRSRSRSRSKTRTPRRSKSKSPSR 180

134

*****::*****:** ************:*** :***: ** **:*: ******* *

SP|Q01130|SRSF2_HUMAN SRSRSRSRSRSRSRS-PPPVSKRESKSRSRSKSPPKSPEEEGAVSS-- 221 SP|Q5R1W5|SRSF2_PANTR SRSRSRSRSRSRSRS-PPPVSKREPKSRSRSKSPPESPEEEGAVSS-- 221 SP|Q62093|SRSF2_MOUSE SRSRSRSRSRSRSRS-PPPVSKRESKSRSRSKSPPKSPEEEGAVSS-- 221 SP|P30352|SRSF2_CHICK SRSRSRSRSRSRSRS-PPPTSKRESNSRSRSKSPPKSPEEEGAVSS-- 221 TR|Q6P366|Q6P366_XENTR SRSRSRSRSRSRTRNSPPPPQNSNSKSRSRSQSPPKSPEEEGAVSS-- 220 TR|Q7ZV13|Q7ZV13_DANRE SRSRSKSKSHSRS---RTPRSNKGSKSRSRSRSRPKSPEATDDAAVES 225 *****:*:*:**: * .: :*****:* *:*** .: SRSF3

SP|P84103|SRSF3_HUMAN ----MHRDSCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDP 56 TR|H2R7D1|H2R7D1_PANTR ----MHRDSCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDP 56 SP|P84104|SRSF3_MOUSE ----MHRDSCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDP 56 TR|E1C8Y9|E1C8Y9_CHICK ----MHRDSCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDP 56 TR|Q7ZWX7|Q7ZWX7_XENLA ----MHRDSCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDL 56 TR|H2L3N2|H2L3N2_ORYLA IPDSIMHRDCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDP 60 : : .**************************************************

SP|P84103|SRSF3_HUMAN RDAADAVRELDGRTLCGCRVRVELSNGEKRSRNRGPPPSWGRRPRD--DYR-RRSPPPRR 113 TR|H2R7D1|H2R7D1_PANTR RDAADAVRELDGRTLCGCRVRVELSNGEKRSRNRGPPPSWGRRPRD--DYR-RRSPPPRR 113 SP|P84104|SRSF3_MOUSE RDAADAVRELDGRTLCGCRVRVELSNGEKRSRNRGPPPSWGRRPRD--DYR-RRSPPPRR 113 TR|E1C8Y9|E1C8Y9_CHICK RDAADAVRELDGRTLCGCRVRVELSNGEKRSRNRGPPPSWGRRPRD--DYR-RRSPPPRR 113 TR|Q7ZWX7|Q7ZWX7_XENLA RDAADAVRELDGRTLCGCRVRVELSNGEKRSRNRGPPPSWNRRPRDDH---RRRSPPPRR 113 TR|H2L3N2|H2L3N2_ORYLA RDATDAVRELDGRTLCGCRVRVELSNGEKRSRSRGAPPSWSRRPRERDDYRRRSSPPPRR 120 ***:****************************.** **** ****: * ******

SP|P84103|SRSF3_HUMAN RSPRRRSFSRSRSRSLSRDRRRERSLSR------ERNHKPSRSFSRSRSRSRSNERK- 164 TR|H2R7D1|H2R7D1_PANTR RSPRRRSFSRSRSRSLSRDRRRERSLSR------ERNHKPSRSFSRSRSRSRSNERK- 164 SP|P84104|SRSF3_MOUSE RSPRRRSFSRSRSRSLSRDRRRERSLSR------ERNHKPSRSFSRSRSRSRSNERK- 164 TR|E1C8Y9|E1C8Y9_CHICK RSPRRRSFSRSRSRSLSRDRRRERSLSR------ERNHKPSRSFSRSRSRSRSNERK- 164 TR|Q7ZWX7|Q7ZWX7_XENLA RYLMHF--SKFYNCLLKLGLRSAAFIYQLHSYWICWFSSIPPPFPFTEPICLCSFNLHAI 171 TR|H2L3N2|H2L3N2_ORYLA RSPRRRSFSRSRSRSFSKDRRRERSLSR------DRNHKPSRSFSRSRSRSRSNDRK- 171 * : *: . :. * : : . * *:. . . * :

SP|P84103|SRSF3_HUMAN ------TR|H2R7D1|H2R7D1_PANTR ------SP|P84104|SRSF3_MOUSE ------TR|E1C8Y9|E1C8Y9_CHICK ------TR|Q7ZWX7|Q7ZWX7_XENLA IFVYLVFFRTSTPQLEENHL 191 TR|H2L3N2|H2L3N2_ORYLA ------

SRSF5

SP|Q13243|SRSF5_HUMAN MSGCRVFIGRLNPAAREKDVERFFKGYGRIRDIDLKRGFGFVEFEDPRDADDAVYELDGK 60 TR|H2Q8I9|H2Q8I9_PANTR MSGCRVFIGRLNPAAREKDVERFFKGYGRIRDIDLKRGFGFVEFEDPRDADDAVYELDGK 60 TR|Q9D8S5|Q9D8S5_MOUSE MSGCRVFIGRLNPAAREKDVERFFKGYGRIRDIDLKRGFGFVEFEDPRDADDAVYELDGK 60 TR|E1BY00|E1BY00_CHICK MSGCRVFIGRLNPAAREKDVERFFKGYGRIRDIDLKRGFGFVEFEDPRDADDAVYELDGK 60 TR|Q6DK94|Q6DK94_XENTR MSGCRVFIGRLNPAAREKDVERFFKGYGRIRDIDLKRGFGFVEFDDPRDADDAVYELDGK 60 TR|H2LZQ0|H2LZQ0_ORYLA MSGSRVFIGRLSPQARERDVEKFFKGYGRIREINLKNGFGFVEFDDHRDADDAVYELNGK 60 ***.*******.* ***:***:*********:*:**.*******:* **********:**

SP|Q13243|SRSF5_HUMAN ELCSERVTIEHARARSRGG---RGRGRYSDRFSSRRPRNDRRNAPPVRTENRLIVENLSS 117 TR|H2Q8I9|H2Q8I9_PANTR ELCSERVTIEHARARSRGG---RGRGRYSDRFSSRRPRNDRRNAPPVRTENRLIVENLSS 117 TR|Q9D8S5|Q9D8S5_MOUSE ELCSERVTIEHARARSRGG---RGRGRYSDRFSSRRPRNDRRNAPPVRTENRLIVENLSS 117 TR|E1BY00|E1BY00_CHICK ELCSERVTIEHARARSR-G---RGRGRYSDRFSSRRPRSDRRSAPPLRTENRLIVENLSS 116 TR|Q6DK94|Q6DK94_XENTR ELCNERVTIEHARLRSRGGPRGLGRGRYNDRFSSRRPRGDR-SAPPIRTENRLIVENLSS 119 TR|H2LZQ0|H2LZQ0_ORYLA ELLSERVTIEHARSRRGRGGGPPGMARFGGGYRQSRNTG-SRYGPPVRTEHRLIVENLSS 119 ** .********* * * * .*: : . * .**:***.*********

SP|Q13243|SRSF5_HUMAN RVSWQDLKDFMRQAGEVTFADAHRPKLNEGVVEFASYGDLKNAIEKLSGKEINGRKIKLI 177 TR|H2Q8I9|H2Q8I9_PANTR RVSWQDLKDFMRQAGEVTFADAHRPKLNEGVVEFASYGDLKNAIEKLSGKEINGRKIKLI 177 TR|Q9D8S5|Q9D8S5_MOUSE RVSWQDLKDFMRQAGEVTFADAHRPKLNEGVVEFASYGDLKNAIEKLSGKEINGRKIKLI 177 TR|E1BY00|E1BY00_CHICK RVSWQDLKDFMRQAGEVTFADAHRPKLNEGVVEFASYSDLKNAIEKLSGKEINGRKIKLI 176 TR|Q6DK94|Q6DK94_XENTR RVSWQDLKDFMRQAGEVTFADAHRPKLNEGVVEFASYSDLKNAIEKLSGKEINGRKIKLI 179 TR|H2LZQ0|H2LZQ0_ORYLA RISWQDLKDLMRKAGEVTFVDAHRPTKNEGVVEFASRSDLKNAISKLDGTELNGRKLKIF 179 *:*******:**:******.*****. ********* .******.**.*.*:****:*::

SP|Q13243|SRSF5_HUMAN EGSKRHSRSRSRSRSRTRSSSRSRSRSRSRSR------KSYSRSRSRSRS--RSRSKS 227

135

TR|H2Q8I9|H2Q8I9_PANTR EGSKRHSRSRSRSRSRTRSSSRSRSRSRSRSR------KSYSRSRSRSRS--RSRSKS 227 TR|Q9D8S5|Q9D8S5_MOUSE EGSKRHSRSRSRSRSRTRSSSRSRSRSRSRRS------KSYSRSRSR--S--RSRSKS 225 TR|E1BY00|E1BY00_CHICK EGSKRHR-SRSRSRSRSRSSSRSRSRSRSRSR------KSYSRSRS------RSRSKS 221 TR|Q6DK94|Q6DK94_XENTR EGNKRHSRSRSRSRSRSRSSSRSRSRSRSRSR------KSYSRSRSRSRTPRSNRSKS 231 TR|H2LZQ0|H2LZQ0_ORYLA EDSRRS-RSRSRSYSRSRSRSRSRSRSRSRSRSVSRTPEKKMSGGGKSAARSPSRSRSRS 238 * .:* ***** **:** ********** * . .:* .**:*

SP|Q13243|SRSF5_HUMAN RSVSRSPVPEKSQKRGSSSRSKSPASVDRQRSRSRSRSRSVDSGN 272 TR|H2Q8I9|H2Q8I9_PANTR RSVSRSPVPEKSQKRGSSSRSKSPASVDRQRSRSRSRSRSVDSGN 272 TR|Q9D8S5|Q9D8S5_MOUSE RSGSRSPVPEKSQKRGSSSRSKSPASVDRQRSRSRSRSRSVDSGN 270 TR|E1BY00|E1BY00_CHICK RSVSRSPMPEKSQKRGSSSRSKSPSSVDRQRSRSR--SRSVDSGN 264 TR|Q6DK94|Q6DK94_XENTR RSVSRSPVPEKSQKSRS--PSKSPASVDRQKSRSRSRSA--DSRN 272 TR|H2LZQ0|H2LZQ0_ORYLA RSGSRSPAQNKQSRSRS------RSQSRSRSRSPSADSKH 272 ** **** :*..: * *.:**** * ** . SRSF6

SP|Q13247|SRSF6_HUMAN CGERVIVEHARGPRRDRDGYSYGSRSGGGG-----YSSRRTSGRDKYGPPVRTEYRLIVE 115 TR|H2QKD5|H2QKD5_PANTR CGERVIVEHARGPRRDRDGYSYGSRSGGGG-----YSSRRTSGRDKYGPPVRTEYRLIVE 114 SP|Q3TWW8|SRSF6_MOUSE CGERVIVEHARGPRRDRDGYSYGSRSGGGG-----YSSRRTSGRDKYGPPVRTEYRLIVE 115 TR|G1MQC0|G1MQC0_MELGA ------RRQSNFISLYNKGGGGGG-----YSSRRQSGRDKYGPPVRTEHRLIVE 43 TR|Q6NVB3|Q6NVB3_XENTR CGERVIVEHARGPRRDRDGYGYGSRSGYR------NQRSGRDKYGPPVRTEFRLIVE 111 TR|H2LEM4|H2LEM4_ORYLA CGERVIVEHARGPRRDRDGYGGGSSWGGGRKSNAMYPSKTRVGRDKYGPPVRTEYRLIVE 120 *:.. . * . ************.*****

SP|Q13247|SRSF6_HUMAN NLSSRCSWQDLKDFMRQAGEVTYADAHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRN 175 TR|H2QKD5|H2QKD5_PANTR NLSSRCSWQDLKDFMRQAGEVTYADAHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRN 174 SP|Q3TWW8|SRSF6_MOUSE NLSSRCSWQDLKDFMRQAGEVTYADAHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRN 175 TR|G1MQC0|G1MQC0_MELGA NLSSRCSWQDLKDFMRQAGEVTYADAHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRK 103 TR|Q6NVB3|Q6NVB3_XENTR NLSSRCSWQDLKDFMRQAGEVTYADAHKERANEGVIEFRSYSDMKRAMEKLDGTEINGRR 171 TR|H2LEM4|H2LEM4_ORYLA NLSSRCSWQDLKDFMRQAGEVTYADAHKERTNQGVIEFRSYSDMKRALDKLDGTDINGRK 180 ******************************:*:**************::*****:****.

SP|Q13247|SRSF6_HUMAN IRLIEDKPRTSHRRSYSGSRSRSRSRRRSRSRS--RRSSR--SRSRSISKSR------223 TR|H2QKD5|H2QKD5_PANTR IRLIEDKPRTSHRRSYSGSRSRSRSRRRSRSRS--RRSSR--SRSRSISKSR------222 SP|Q3TWW8|SRSF6_MOUSE IRLIEDKPRTSHRRSYSGSRSRSRSRRRSRSRS--RRSSR--SRSRSISKSR------223 TR|G1MQC0|G1MQC0_MELGA IRLVEDKPRSSHRRSYSCSRSRSRSRRRSRSRS--RRSRSSRSRSRSVSKSRSR------155 TR|Q6NVB3|Q6NVB3_XENTR IRLVEGKAR--HRRSYSGSRSRSRSRSRRRSRSRSRQPSHSRSRSRSHSPAKKGRSPAKK 229 TR|H2LEM4|H2LEM4_ORYLA IRLVEDRPH--KRRSYSGSRSRSRSRRRSRSGS--RRSSKSRSRSHSRSRSRSN------230 ***:* : : :***** ******** * ** * *: ***:* * ::

136

:: . : .:* .* : .

SRSF7

SP|Q16629|SRSF7_HUMAN SYYGPLRTVWIARNPPGFAFVEFEDPRDAEDAVRGLDGKVICGSRVRVELSTGMPRRSRF 91 TR|H2QHS3|H2QHS3_PANTR SYYGPLRTVWIARNPPGFAFVEFEDPRDAEDAVRGLDGKVICGSRVRVELSTGMPRRSRF 91 SP|Q8BL97|SRSF7_MOUSE SYYGPLRTVWIARNPPGFAFVEFEDPRDAEDAVRGLDGKVICGSRVRVELSTGMPRRSRF 120 TR|Q5ZMI0|Q5ZMI0_CHICK SYYGPLRTVWIARNPPGFAFVEFEDPRDAEDAVLGLDGKIICGSRVRVEVSTGMPRRSRY 89 TR|Q6AZT1|Q6AZT1_XENLA SYYGPLRTVWIARNPPGFAFVEFEDTRDAEDAVRGLDGKVICGSRVRVELSTGMPRRSRY 91 TR|A3KNI1|A3KNI1_DANRE GYYGPLRSVWIARNPAGFAFVEFEDPRDAEDSVRGLDGKVICGSRVRVELSTGMPRRSRY 91 .******:******* ********* *****:* *****:*********:*********:

SP|Q16629|SRSF7_HUMAN DRPPARRPFDPNDRCYECGEKGHYAYDCHRYSRRRRSRSRSRSHSRSRGRRYSRSRSRSR 151 TR|H2QHS3|H2QHS3_PANTR DRPPARRPFDPNDRCYECGEKGHYAYDCHRYSRRRRSRSRSRSHSRSRGRRYSRSRSRSR 151 SP|Q8BL97|SRSF7_MOUSE DRPPARRPFDPNDRCYECGEKGHYAYDCHRYSRRRRSRSRSRSHSRSRGRRYSRSRSRSR 180 TR|Q5ZMI0|Q5ZMI0_CHICK DRPPARRPFDPNDRCYECGEKGHYAYDCHRYSRRRRSRSRSRSRSRSRGRRYSRSRSRSR 149 TR|Q6AZT1|Q6AZT1_XENLA DRPPARRPFDPSDRCYECGEKGHYAYDCQRYSRRRRSRTRSRSHSRSRGRRYSRSRSRSR 151 TR|A3KNI1|A3KNI1_DANRE DHPPSRRPFDPNDRCYECGEKGHYAYDCHRYSRRRRTRSRSRS--HSRGRRYGRSHSRSR 149 *:**:******.****************:*******:*:**** :******.**:****

SRSF9

137

SP|Q13242|SRSF9_HUMAN -MSGWADE--RGGEGDGRIYVGNLPTDVREKDLEDLFYKYGRIREIELKNRHG--LVPFA 55 TR|H2Q712|H2Q712_PANTR -MSGWADE--RGGEGDGRIYVGNLPTDVREKDLEDLFYKYGRIREIELKNRHG--LVPFA 55 SP|Q9D0B0|SRSF9_MOUSE MSSGWADE--RGGEGDGRIYVGNLPSDVREKDLEDLFYKYGRIREIELKNRHG--LVPFA 56 TR|Q6GLG3|Q6GLG3_XENTR -MSGWDREAARTGSGDGRIYVGNLPADIREKELEDLFDRYGRIRTIELKNRGGSSAAPFA 59 TR|Q6NXE0|Q6NXE0_DANRE ------MSDGRIYVGNLPMDVQERDIEDLFFKYGKIRDIELKNNRS--TIPFA 45 .********** *::*:::**** :**:** *****. . ***

SP|Q13242|SRSF9_HUMAN FVRFEDPRDAEDAIYGRNGYDYGQCRLRVEFPRTYGG-----RGGWPR--GGRNGPPTRR 108 TR|H2Q712|H2Q712_PANTR FVRFEDPRDAEDAIYGRNGYDYGQCRLRVEFPRTYGG-----RGGWPR--GGRNGPPTRR 108 SP|Q9D0B0|SRSF9_MOUSE FVRFEDPRDAEDAIYGRNGYDYGQCRLRVEFPRTYGG-----RGGWPR--GARNGPPTRR 109 TR|Q6GLG3|Q6GLG3_XENTR FISFQDPRDAEDAVFARNGYEFGSCRLRVEFPRSFRG-----SGGGYGGSRGRNGPPSRR 114 TR|Q6NXE0|Q6NXE0_DANRE FVRFEDPRDAEDAVFGRNGYGFGDCKLRVEYPRSSGSKFSGPAGGGGGGPRGRFGPPTRR 105 *: *:********::.**** :*.*:****:**: . ** .* ***:**

SP|Q13242|SRSF9_HUMAN SDFRVLVSGLPPSGSWQDLKDHMREAGDVCYADVQKDGVGMVEYLRKEDMEYALRKLDDT 168 TR|H2Q712|H2Q712_PANTR SDFRVLVSGLPPSGSWQDLKDHMREAGDVCYADVQKDGVGMVEYLRKEDMEYALRKLDDT 168 SP|Q9D0B0|SRSF9_MOUSE SDFRVLVSGLPPSGSWQDLKDHMREAGDVCYADVQKDGMGMVEYLRKEDMEYALRKLDDT 169 TR|Q6GLG3|Q6GLG3_XENTR SEYRVIVSGLPPSGSWQDLKDHMREAGDVCYADVHKDGMGIVEFIRKEDMEYALRKLDDT 174 TR|Q6NXE0|Q6NXE0_DANRE SEFRVIVTGLPPTGSWQDLKDHMREAGDVCFADVQRDGEGVVEFLRREDMEYALRRLDST 165 *::**:*:****:*****************:***::** *:**::*:********:**.*

SP|Q13242|SRSF9_HUMAN KFRSHEGETSYIRVYPERSTSYGYSRSRSGSRGRDS-PYQSRGSPH--YFSPFRPY---- 221 TR|H2Q712|H2Q712_PANTR KFRSHEGETSYIRVYPERSTSYGYSRSRSGSRGRDS-PYQSRGSPH--YFSPFRPY---- 221 SP|Q9D0B0|SRSF9_MOUSE KFRSHEGETSYIRVYPERSTSYGYSRSRSGSRGRDS-PYQSRGSPH--YFSPFRPY---- 222 TR|Q6GLG3|Q6GLG3_XENTR KFRSHEGETSYIRVCPERNTSY--SRSRSRSRGRDS-PYQSRRSPR--YASPFRPY---- 225 TR|Q6NXE0|Q6NXE0_DANRE EFRSHQGETAYIRVMEERGTSWGRSRSRSRSRGRYTPPYQSRGSPPPRYRSPPRHMTRHS 225 :****:***:**** ** **: ***** **** : ***** ** * ** *

SRSF10

SP|O75494|SRS10_HUMAN MSRYLRPPNTSLFVRNVADDTRSEDLRREFGRYGPIVDVYVPLDFYTRRPRGFAYVQFED 60 TR|H2R9C4|H2R9C4_PANTR MSRYLRPPNTSLFVRNVADDTRSEDLRREFGRYGPIVDVYVPLDFYTRRPRGFAYVQFED 60 SP|Q9R0U0|SRS10_MOUSE MSRYLRPPNTSLFVRNVADDTRSEDLRREFGRYGPIVDVYVPLDFYTRRPRGFAYVQFED 60 TR|Q5ZMR4|Q5ZMR4_CHICK MSRYLRPPNTSLFVRNVADDTRSEDLRREFGRYGPIVDVYVPLDFYTRRPRGFAYVQFED 60 TR|Q5XGF2|Q5XGF2_XENTR MSRYLRPPNTSLFVRNIADDIRSEDLRREFGRYGPIVDVYVPLDYYTRRPRGFAYVQFED 60 TR|H2LU61|H2LU61_ORYLA MARYMRPPNTSLFVRNISDESRPEDLRREFGRYGPIVDVYIPLDFYTRQPRGFAYIQFED 60 *:**:***********::*: * *****************:***:***:******:****

SP|O75494|SRS10_HUMAN VRDAEDALHNLDRKWICGRQIEIQFAQGDRKTPNQMKAKEGRNVYSSSRYDDYDRYRRSR 120 TR|H2R9C4|H2R9C4_PANTR VRDAEDALHNLDRKWICGRQIEIQFAQGDRKTPNQMKAKEGRNVYSSSRYDDYDRYRRSR 120 SP|Q9R0U0|SRS10_MOUSE VRDAEDALHNLDRKWICGRQIEIQFAQGDRKTPNQMKAKEGRNVYSSSRYDDYDRYRRSR 120 TR|Q5ZMR4|Q5ZMR4_CHICK VRDAEDALHNLDRKWICGRQIEIQFAQGDRKTPNQMKAKEGRNLYSSSRYDDYDRYRRSR 120 TR|Q5XGF2|Q5XGF2_XENTR VRDAEDALHNLDKKWICGRQIEIQFAQGDRKTPNQMKAKEGRSTYGSSRYDDDRHNRRSR 120 TR|H2LU61|H2LU61_ORYLA VRDAEDALHSLDRKWVCGRQIEIQFAQGDRKTPNQMKTKERRPRADHLDMTTTTEIADAD 120 *********.**:**:*********************:** * . :

SP|O75494|SRS10_HUMAN SRSKSQPKKEMKAKS--RSRSASHTKTRGTSKTDSKTHYKSGSRYEKESRKKEPPRSKSQ 238 TR|H2R9C4|H2R9C4_PANTR SRSKSQPKKEMKAKS--RSRSASHTKTRGTSKTDSKTHYKSGSRYEKESRKKEPPRSKSQ 238 SP|Q9R0U0|SRS10_MOUSE SRSKSQPKKEMKAKS--RSRSASHTKTRGTSKTDSKTHYKSGSRYEKESRKKEPPRSKSQ 238 TR|Q5ZMR4|Q5ZMR4_CHICK SRSKSQPKKEMKAKS--RSRSASHTKSRGTSKTDSKTHYKSSSRYEKESRKKEPARSKSQ 238 TR|Q5XGF2|Q5XGF2_XENTR SRSKSEPKKTVREQ-----RSGSRSHSRGHSKADSKSRCRENSRYNRESRRDEHEQSKSP 234 TR|H2LU61|H2LU61_ORYLA ------DECIQEEEAGVARKTDTSTNLGESPERGLDH------AKDLHPLTKTS 202 .: :: : *. . : . * * . : :. :*:

SP|O75494|SRS10_HUMAN SRSQSRSRSKSRSRSWTSPKSSGH 262

138

SRSF11

SP|Q05519|SRS11_HUMAN ELRLFPPDDSPLPVSSRVCFVKFHDPDSAVVAQHLTNTVFVDRALIVVPYAEGVIPDEAK 120 TR|H2R8Z8|H2R8Z8_PANTR ELRLFPPDDSPLPVSSRVCFVKFHDPDSAVVAQHLTNTVFVDRALIVVPYAEGVIPDEAK 120 TR|Q3UIX4|Q3UIX4_MOUSE ELRLFPPDDSPLPVSSRVCFVKFHDPDSAVVAQHLTNTVFVDRALIVVPYAEGVIPDETK 113 TR|F1NWC7|F1NWC7_CHICK ------DSPLPVSSRVCFVKFHDPDSAVVAQHLTNTVFVDRALIVVPYAEGVIPDETK 52 TR|F7BMD3|F7BMD3_XENTR ELRLFPPDDSPLPVTSRVCFVKFQDPDSAVVAQHLTNTVFVDRALIVVPYAEGIIPDEAK 93 TR|Q803U8|Q803U8_DANRE ELRLFPPDDSPLPVTSRVCFVKFHEPESVGVSQHLTNTVFVDRALIVVPFAEGVIPDESK 94 ******:********::*:*. *:*****************:***:****:*

SP|Q05519|SRS11_HUMAN ALSLLAPANAVAGLLPGGGLLPTPNPLTQIGAVPLAALGAPTLDPALAALGLPGANLNSQ 180 TR|H2R8Z8|H2R8Z8_PANTR ALSLLAPANAVAGLLPGGGLLPTPNPLTQIGAVPLAALGAPTLDPALAALGLPGANLNSQ 180 TR|Q3UIX4|Q3UIX4_MOUSE ALSLLAPANAVAGLLPGGGLLPTPNPLTQIGAVPLAALGAPALDPALAALGLPGTNLNSQ 173 TR|F1NWC7|F1NWC7_CHICK ALSLLAPANAVAGLLPGGGLLPTPNPLSQIGAVPLAALGAPALDPALAALGLPGANLNSQ 112 TR|F7BMD3|F7BMD3_XENTR ALSLVAPANAVAGLLPGGGLLPTPNPLSQIGAVPLAALGAPTLDPTLAALTLPGANLNSQ 153 TR|Q803U8|Q803U8_DANRE AMSLLAPANAVAGLLPGGGLLPTPNPVPSIGGVPLGGLGGPNLDPM-AALAMAAPNINPQ 153 *:**:*********************: .**.***..**.* *** *** : . *:* *

SP|Q05519|SRS11_HUMAN SLAADQLLKLMSTVDPKLNHVAAGL-VSPSLKSDTSSKEIEEAMKRVREAQSLISAAIEP 239 TR|H2R8Z8|H2R8Z8_PANTR SLAADQLLKLMSTVDPKLNHVAAGL-VSPSLKSDTSSKEIEEAMKRVREAQSLISAAIEP 239 TR|Q3UIX4|Q3UIX4_MOUSE SLAADQLLKLMSTVDPKLNHVAAGL-VSPSLKSDTSSKEIEEAMKRVREAQSLISAAIEP 232 TR|F1NWC7|F1NWC7_CHICK SLAADQLLKLMSTVDPKLNHVAAGL-VSPSLKSDTSSKEIEEAMKRVREAQSLISAAIEP 171 TR|F7BMD3|F7BMD3_XENTR SLAADQLLKLMSTVDPKLNHVTAGL-VSPSLKSDTSSKDIEEAMKRVREAQSLISAAIEP 212 TR|Q803U8|Q803U8_DANRE SLSAEQLMKLMASIDPKLNPLAAGLNLTPGLKADASNKEIEEAMKRVREAQSLISAAIEP 213 **:*:**:***:::***** ::*** ::*.**:*:*.*:*********************

SP|Q05519|SRS11_HUMAN DKKEEK-RRHSRSRSRSRRRRTPSSSRHRRSRSRSRRRSHSKSRSRRRSKSPRRRRSHSR 298 TR|H2R8Z8|H2R8Z8_PANTR DKKEEK-RRHSRSRSRSRRRRTPSSSRHRRSRSRSRRRSHSKSRSRRRSKSPRRRRSHSR 298 TR|Q3UIX4|Q3UIX4_MOUSE DKKEEK-RRHSRSRSRSRRRRTPSSSRHRRSRSRSRRRSHSKSRSRRRSKSPRRRRSHSR 291 TR|F1NWC7|F1NWC7_CHICK DKKDEK-RRHSRSRSRSRRRRTPSSSRHRRSRSRSRRRSHSKSRSRRRSKSPRRRRSHSR 230 TR|F7BMD3|F7BMD3_XENTR DKKDEKSKKHSRSRSHSRRRRTPSSSRHRRSRSRSRRRSHSKSRSRRRSKSPRRRRSHSR 272 TR|Q803U8|Q803U8_DANRE GSKKDDKRKRSRSRSRSRRRRSRSRSRHRRSKSHSRRRS--RSRSRRRSKSPRRRRSHSR 271 .*.:. :::*****:*****: * ******:*:***** :******************

SP|Q05519|SRS11_HUMAN ERGRRSRSTSKTRDKKKEDKEKKRSKTPPKSYSTARRSRSASRERRRRRSRSGTRSPKKP 358 TR|H2R8Z8|H2R8Z8_PANTR ERGRRSRSTSKTRDKKKEDKEKKRSKTPPKSYSTARRSRSASRERRRRRSRSGTRSPKKP 358 TR|Q3UIX4|Q3UIX4_MOUSE ERGRRSRSTSKARDKKKEDKEKKRSKTPPKSYSTARRSRSASRERRRRRSRSGTRSPKKP 351 TR|F1NWC7|F1NWC7_CHICK ERSRRSRSTSKTRDKKKEEKEKKRSKTPPKSYSTTRRSRSTSRERRRRRSRSGTRSPKKP 290 TR|F7BMD3|F7BMD3_XENTR ERSRRSRSTSKPREKKREEKEKKRSKTPPKSYSTTRRSRSTSRDKRRRKSRSGSRSPKKL 332 TR|Q803U8|Q803U8_DANRE DRSRRSRSR----DRRKDEKYRKRSKTPPKSYSSARRSRSTSRKR-HRRSRSMSRSPKKS 326 :*.***** ::::::* :***********::*****:**.: :*:*** :*****

SP|Q05519|SRS11_HUMAN RSPKRKLSRSPSPRRHKKEKKKDKDKERSRDERERST------SKK--KKSKDKEKDR 408 TR|H2R8Z8|H2R8Z8_PANTR RSPKRKLSRSPSPRRHKKEKKKDKDKERSRDERERST------SKK--KKSKDKEKDR 408 TR|Q3UIX4|Q3UIX4_MOUSE RSPKRKLSRSPSPRRHKKEKKKDKDKERSRDERERST------SKK--KRSKDKEKER 401 TR|F1NWC7|F1NWC7_CHICK RSPKRKMSRSPSPRRHKKEKKKDKDKERSRDERERST------SKK--KKSKDKEKDR 340 TR|F7BMD3|F7BMD3_XENTR RSPKRKPSRSPSPRRRFNPKTDHKYKAFALLYKISVV------ANKQQMNYRLKYLWK 384 TR|Q803U8|Q803U8_DANRE RSPKRKLSRSPSPRRHKKEKKKDKDRERDRDRDRKEDRDRNREKRERSTSKKSKDKDKDR 386 ****** ********: : *...* : .. . : * :

139

TR|Q803U8|Q803U8_DANRE DRKSDSEKGDVKVTRDYDEEEQGYDSEHEEHERNSDA-ASSPHAKEPLADSADDAGRGES 445 .*.. ::* ***************:*::*. : * .:. .: :*

TRA2A

SP|Q13595|TRA2A_HUMAN MSDVEENNFEGRESRSQSKSPTGTPARVKSESRSGSRSPSRVSKHSESHSRSRSKSRSRS 60 TR|H2QU97|H2QU97_PANTR MSDVEENNFEGRESRSQSKSPTGTPARVKSESRSGSRSPSRVSKHSESHSRSRSKSRSRS 60 SP|Q6PFR5|TRA2A_MOUSE MSDVEENNFEGRESRSQSKSPTGTPARVKSESRSGSRSPSRVSKHSESHSRSRSKSRSRS 60 TR|F1NPM7|F1NPM7_CHICK MSDVEENNFEGRESRSQSKSPAGSPARVKSESRSGSRSPSRASKHSESHSRSRSKSRSRS 60 TR|F6YWF8|F6YWF8_XENTR NGRVFSKNIEDKESRSRSKSPAESAPRVKSESRSRSRSASRASKRSESRSRSRSKSRSRS 60 TR|Q7T2A0|Q7T2A0_DANRE MSDTEEQQFQRRESRSASKSDRGSPAQPKMESRSGSPSPSRASKRSDSRSRSRSKSRSRS 60 . . .:::: :**** *** : : * **** * * **.**:*:*:***********

SP|Q13595|TRA2A_HUMAN RRHSHRRYTRSRSHS--HSHRRRSRSRSYTPEYRRRRSRSHSPMSNRRRHTGS------111 TR|H2QU97|H2QU97_PANTR RRHSHRRYTRSRSHS--HSHRRRSRSRSYTPEYRRRRSRSHSPMSNRRRHTGS------111 SP|Q6PFR5|TRA2A_MOUSE RRHSHRRYTRSRSH----SHRRRSRSRSYTPEYRRRRSRSHSPMSNRRRHTGS------109 TR|F1NPM7|F1NPM7_CHICK RRHSHRRYTRSRSHSHSHSHRRRSRSRSYTPEYRRRRSRSHSPMSNRRRHTGS------113 TR|F6YWF8|F6YWF8_XENTR RRHSHRRYSRSRSRS--HSRKRRSKSRSYTPEYRRRRSRSHSPMSNRRRHNGS------111 TR|Q7T2A0|Q7T2A0_DANRE RRHSNRRYSRSRSH----SHRKKSRSRSYSPESRRRRSRSASPNSNRRKHAGSRSSYSHD 116 ****.***:****: *::::*:****:** ******* ** ****:* **

SP|Q13595|TRA2A_HUMAN ------RANPDPNTCLGVFGLSLYTTERDLREVFSRYGPLSGVNVVYDQRTGRSR 160 TR|H2QU97|H2QU97_PANTR ------RANPDPNTCLGVFGLSLYTTERDLREVFSRYGPLSGVNVVYDQRTGRSR 160 SP|Q6PFR5|TRA2A_MOUSE ------RANPDPNTCLGVFGLSLYTTERDLREVFSRYGPLSGVNVVYDQRTGRSR 158 TR|F1NPM7|F1NPM7_CHICK ------RANPDPNTCLGVFGLSLYTTERDLREVFSRYGPLTGVNVVYDQRTGRSR 162 TR|F6YWF8|F6YWF8_XENTR ------RANPDPNICIGVFGLSLYTTERDLREVFSRYGPLSGVNVVYDQRTGRSR 160 TR|Q7T2A0|Q7T2A0_DANRE SKKDSHNQGDARANPDPNTCLGVFGLSLYTTERDLREVFSRYGSLAGVNVVYDQRTGRSR 176 ******* *:********************** *:**************

SP|Q13595|TRA2A_HUMAN GFAFVYFERIDDSKEAMERANGMELDGRRIRVDYSITKRAHTPTPGIYMGRPTHSGGGGG 220 TR|H2QU97|H2QU97_PANTR GFAFVYFERIDDSKEAMERANGMELDGRRIRVDYSITKRAHTPTPGIYMGRPTHSGGGGG 220 SP|Q6PFR5|TRA2A_MOUSE GFAFVYFERIDDSKEAMERANGMELDGRRIRVDYSITKRAHTPTPGIYMGRPTHSGGGGG 218 TR|F1NPM7|F1NPM7_CHICK GFAFVYFERIDDSKEAMEHANGMELDGRRIRVDYSITKRAHTPTPGIYMGRPTHSGGGGG 222 TR|F6YWF8|F6YWF8_XENTR GFAFVYFERIEDSREAMEHANGMELDGRRIRVDYSITKRAHTPTPGIYMGRPTQLGVGLK 220 TR|Q7T2A0|Q7T2A0_DANRE GFAFVYFEHIDDAKEAMERANGMELDGRRIRVDYSITKRPHTPTPGIYMGRPTHNGGGGG 236 ********:*:*::****:******************** *************: * *

SP|Q13595|TRA2A_HUMAN GGG---GGGGGGGGRRRDSYYDRGYDRGYDR-YEDYDYRYR-RRSPSPYYSRYRSRSRSR 275 TR|H2QU97|H2QU97_PANTR GGG---GGGGGGGGRRRDSYYDRGYDRGYDR-YEDYDYRYR-RRSPSPYYSRYRSRSRSR 275 SP|Q6PFR5|TRA2A_MOUSE GGGGGGGGGGGGGGRRRDSYYDRGYDRGYDR-YEDYDY--R-RRSPSPYYSRYRSRSRSR 274 TR|F1NPM7|F1NPM7_CHICK ------GGAGRRRDSYYDRGYDRGYDR-YEEYDYRYR-RRSPSPYYSRYRSRSRSR 270 TR|F6YWF8|F6YWF8_XENTR GGEHINA---GLSSRRRDSYYDRGYDRGYDR-YDEYDYRYR-RRSPSPYYSRYRSRSRSR 275 TR|Q7T2A0|Q7T2A0_DANRE GGGS------SSGGRRRDSYYDRGYDRGYDRGYDEYDYRYSRRRSPSPYYSRYRSRSRSR 290 . ..***************** *::*** ******************

TRA2B

140

...*::: :*********.* : *.**. :: *:*:.:. :*:*::****.:

SP|P62995|TRA2B_HUMAN RSRSRRSSRRHYTRSRSRSRSH-RRSRSRSYSRDYR-RRHSHSHSPMSTRRRHVGNRANP 114 TR|H2QNW6|H2QNW6_PANTR RSRSRRSSRRHYTRSRSRSRSH-RRSRSRSYSRDYR-RRHSHSHSPMSTRRRHVGNRANP 114 SP|P62996|TRA2B_MOUSE RSRSRRSSRRHYTRSRSRSRSH-RRSRSRSYSRDYR-RRHSHSHSPMSTRRRHVGNRANP 114 TR|Q9DDU8|Q9DDU8_CHICK RSRSRRSSRRHYTRSRSRSRSH-RRSRSRSYSRDYR-RRHSHSHSPMSTRRRHIGNRANP 114 TR|Q6DES6|Q6DES6_XENTR RSRSRRSSRRHYTRSRTRSRSR-RRSRSRSHSRDYR-RRRSHSHSPMSTRRRHVGNRANP 118 TR|Q7ZUG9|Q7ZUG9_DANRE RSRSHRSSRRHYSRSRSRSYSRRRRSRSRSYSSEYHRRRSSHSHSPMSNRRRHIGDRANP 119 ****:*******:***:** *: *******:* :*: ** ********.****:*:****

SP|P62995|TRA2B_HUMAN DPNCCLGVFGLSLYTTERDLREVFSKYGPIADVSIVYDQQSRRSRGFAFVYFENVDDAKE 174 TR|H2QNW6|H2QNW6_PANTR DPNCCLGVFGLSLYTTERDLREVFSKYGPIADVSIVYDQQSRRSRGFAFVYFENVDDAKE 174 SP|P62996|TRA2B_MOUSE DPNCCLGVFGLSLYTTERDLREVFSKYGPIADVSIVYDQQSRRSRGFAFVYFENVDDAKE 174 TR|Q9DDU8|Q9DDU8_CHICK DPNCCLGVFGLSLYTTERDLREVFSKYGPIADVSIVYDQQSRRSRGFAFVYFENVEDAKE 174 TR|Q6DES6|Q6DES6_XENTR DPNCCLGVFGLSLYTTERDLREVFSKYGPISDVSIVYDQQSRRSRGFSFVYFENVDDAKE 178 TR|Q7ZUG9|Q7ZUG9_DANRE DPNCCLGVFGLSLYTTERDLREVFSKYGPLSDVCIVYDQQSRRSRGFALVYFENREDSKE 179 *****************************::**.*************::***** :*:**

SP|P62995|TRA2B_HUMAN AKERANGMELDGRRIRVDFSITKRPHTPTPGIYMGRPTYGS----SRRRDYYDRGYDR-G 229 TR|H2QNW6|H2QNW6_PANTR AKERANGMELDGRRIRVDFSITKRPHTPTPGIYMGRPTYGS----SRRRDYYDRGYDR-G 229 SP|P62996|TRA2B_MOUSE AKERANGMELDGRRIRVDFSITKRPHTPTPGIYMGRPTYGS----SRRRDYYDRGYDR-G 229 TR|Q9DDU8|Q9DDU8_CHICK AKERANGMELDGRRIRVDFSITKRPHTPTPGIYMGRPTYGS----SRRRDYYDRGYDR-G 229 TR|Q6DES6|Q6DES6_XENTR AKERANGMELDGRRIRVDFSITKRPHTPTPGIYMGRPTYGS----SRRRDYYDRGYDRGG 234 TR|Q7ZUG9|Q7ZUG9_DANRE AKERANGMELDGRRIRVDYSITKGPHTPTPGIYMGRPTYGGGPSVSRRRDSYDRGYERGY 239 ******************:**** ****************. ***** *****:*

141

APPENDIX D – Raw SpliceAid 2 Data

Sequences were searched in sections. Section of sequences are given and the nt position provided according to Flt1 genomic sequence, NM002019. Each section is followed by the raw data returns from Spliceaid 2 [1]: position, protein name, recognized sequence, protein notes, PubMed ID, reference, article notes, gene/construct (Target RNA), splicing assay and binding assay.

sFlt1_v1

Exon 13 (134300 – 134608)

CTCTGATTGTAATTTCTTTCTTCTGGAGGATTTCTTCCCCTGTGTATACATTCCTGGCTCTGCAGGCATAGGTGCCTG AATCTTGCAGGGAAACATTCATGATGGTAAGATTAAGAGTGATGGAGTGCTCCTTAGTGATGGCCATTTTTTGCTT GCTAATACTGTAGTGCATTGTTCTGTTATTAACTGTCCGCAGTAAAATCCAAGTAACGTCTCTGTATAAGAACTTGT TAACTGTGCAAGACAGTTTCAGGTCCTCTCCTTCCGTCGGCATTTTTTCCAAGTTAACATGAAACCCATTTGGCACA T

Gene/Constru Protein Recognized Position Protein Notes PubMed ID Reference Article notes ct (Target Splicing Assay Binding Assay Name Sequence RNA)

Rooke N, Markovtsov V, Cagavi E, Gene Name and Black DL. Synonymous: SFRS2, (2003) In vitro splicing factor Roles for SR splicing with UV crosslink and arginine/serine-rich 2, SC-35, Construct of c- proteins and Weri-1, Weri-1 immunoprecipitation with 25-30 SC35 GGAGGA SFRS2A, SRp30b, PR264. 12612063 src [20779] hnRNP A1 in S100 and HeLa Weri-1 and HeLa nuclear SC35 accelerates EX_N1. the regulation nuclear extracts. transcriptional elongation (co- of c-src exon extracts. transcriptional splicing) N1. (PMID: 18641664). Mol Cell Biol. 23(6):1874- 1884.

Cavaloc Y, Bourgeois Gene Name CF, Kister L, and Stevenin J. Synonymous: Sequences of 20 nt random for (1999) SELEX of 20-nt random with SFRS3, SELEX. Constructs of The splicing recombinant protein. Winners splicing factor EXE1A_Adenovirus - In vitro factors 9G8 confirmed by EMSA with arginine/serine- partial_INT_E1A_Adenovirus - splicing SRp2 and SRp20 recombinant protein, UV 93-100 ACAUUCAU rich 3. 10094314 partial_INT_FN1 - EX_ED1_FN1 in HeLa 0 transactivate crosslink, complementation The shuttling of FIBRONECTIN (FN1) [2335] S100 splicing assay, immunoprecipitations protein SRp20 for in vitro splicing. Construct of extracts. through with HeLa S100 and nuclear binds TAP and Sp1 unit of Adenovirus E1A for in different and extracts. can function as vitro splicing. specific export factors enhancers. (18364396). RNA. 5(3): 468-483.

Liu HX, Zhang Construct of SELEX imposing a M, Krainer AR. EX_M1 - selection of the (1998) INT1 - constructs for Gene Name and Identification of EX_M2 splicing rather than Synonymous: functional murine IgM for binding. UV crosslink, competition and SFRS5, splicing 22-26 SRp40 UCUGG 9649504 exonic splicing and its Selection of the immunoprecipitation assays in HeLa factor enhancer motifs variants constructs by nuclear extracts. arginine/serine- recognized by obtained by splicing in HeLa rich 5, HRS. individual SR replacing the S100 extracts proteins. natural ESE in complemented by Genes Dev. the M2 exon recombinant SR

142

12(13): 1998- with 20nt protein. Winners 2012. random. are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the Liu HX, Zhang Construct of constructs for M, Krainer AR. EX_M1 - splicing rather than (1998) INT1 - for binding. Identification of EX_M2 Gene Name and Selection of the functional murine IgM Synonymous: constructs by exonic splicing and its UV crosslink, competition and SFRS5, splicing splicing in HeLa 62-66 SRp40 GCAGG 9649504 enhancer motifs variants immunoprecipitation assays in HeLa factor S100 extracts recognized by obtained by nuclear extracts. arginine/serine- complemented by individual SR replacing the rich 5, HRS. recombinant SR proteins. natural ESE in protein. Winners Genes Dev. the M2 exon are confirmed by in 12(13): 1998- with 20nt vitro splicing in 2012. random. HeLa nuclear extracts.

SELEX imposing a selection of the Liu HX, Zhang Construct of constructs for M, Krainer AR. EX_M1 - splicing rather than (1998) INT1 - for binding. Identification of EX_M2 Gene Name and Selection of the functional murine IgM Synonymous: constructs by exonic splicing and its UV crosslink, competition and SFRS5, splicing splicing in HeLa 85-89 SRp40 GCAGG 9649504 enhancer motifs variants immunoprecipitation assays in HeLa factor S100 extracts recognized by obtained by nuclear extracts. arginine/serine- complemented by individual SR replacing the rich 5, HRS. recombinant SR proteins. natural ESE in protein. Winners Genes Dev. the M2 exon are confirmed by in 12(13): 1998- with 20nt vitro splicing in 2012. random. HeLa nuclear extracts.

SELEX imposing a selection of the Liu HX, Zhang Construct of constructs for M, Krainer AR. EX_M1 - splicing rather than (1998) INT1 - for binding. Identification of EX_M2 Gene Name and Selection of the functional murine IgM Synonymous: constructs by exonic splicing and its UV crosslink, competition and SFRS5, splicing splicing in HeLa 190-194 SRp40 UCCGC 9649504 enhancer motifs variants immunoprecipitation assays in HeLa factor S100 extracts recognized by obtained by nuclear extracts. arginine/serine- complemented by individual SR replacing the rich 5, HRS. recombinant SR proteins. natural ESE in protein. Winners Genes Dev. the M2 exon are confirmed by in 12(13): 1998- with 20nt vitro splicing in 2012. random. HeLa nuclear extracts.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Chabot In vitro B. (2007) Gene Name and splicing with SELEX of 20nt random with hnRNP Synthesized oligos. SRp3 Synonymous: SFRS9, HeLa recombinant protein. EMSA, UV AGGAU 17548433 I/PTB can Sequences of 20nt random 27-31 0c splicing factor nuclear crosslink, SDS-PAGE with HeLa antagoniz for SELEX. arginine/serine-rich 9. extracts, nuclear extracts. e the siRNA. splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

SRp3 Gene Name and Paradis C, Synthesized oligos. In vitro SELEX of 20nt random with AGGCA 17548433 64-68 0c Synonymous: SFRS9, Cloutier Sequences of 20nt random splicing with recombinant protein. EMSA, UV

143

splicing factor P, Shkreta for SELEX. HeLa crosslink, SDS-PAGE with HeLa arginine/serine-rich 9. L, nuclear nuclear extracts. Toutant J, extracts, Klarskov siRNA. K, Chabot B. (2007) hnRNP I/PTB can antagoniz e the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Chabot In vitro B. (2007) Gene Name and splicing with SELEX of 20nt random with hnRNP Synthesized oligos. 223- SRp3 Synonymous: SFRS9, HeLa recombinant protein. EMSA, UV AGAAC 17548433 I/PTB can Sequences of 20nt random 0c splicing factor nuclear crosslink, SDS-PAGE with HeLa 227 antagoniz for SELEX. arginine/serine-rich 9. extracts, nuclear extracts. e the siRNA. splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Chabot In vitro B. (2007) Gene Name and splicing with SELEX of 20nt random with hnRNP Synthesized oligos. 241- SRp3 Synonymous: SFRS9, HeLa recombinant protein. EMSA, UV AAGAC 17548433 I/PTB can Sequences of 20nt random 0c splicing factor nuclear crosslink, SDS-PAGE with HeLa 245 antagoniz for SELEX. arginine/serine-rich 9. extracts, nuclear extracts. e the siRNA. splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Tacke R, Tohyama M, Ogawa S, Manley JL. (1998) Human Sequences of 20 nt random for SELEX of 20nt random with Gene Name and Tra2 SELEX. Sequence of beta-globin In vitro splicing in recombinant protein, confirmed by HTra2al Synonymous: proteins 222-226 AAGAA 9546399 [3043] and constructs of murine HeLa S100 and EMSA in HeLa nuclear extract and pha TRA2A, transformer-2 are IgM-based pre-mRNA for in vitro nuclear extracts. S100. EMSA with recombinant alpha, HSU53209. sequence- splicing. protein. specific activators of pre- mRNA splicing. Cell. 93(1): 139-148.

144

Tacke R, Tohyama M, Ogawa S, Manley JL. (1998) Gene Name and Synonymous: Sequences of 20 nt Human SFRS10, splicing factor random for SELEX. In vitro Tra2 SELEX of 20nt random with arginine/serine-rich 10 954 Sequence of beta- splicing in HTra2 proteins recombinant protein, confirmed by 222-226 AAGAA (transformer 2 homolog, 639 globin [3043] and HeLa S100 beta1 are EMSA in HeLa nuclear extract and Drosophila), TRA2B, SRFS10, 9 constructs of murine and nuclear sequence- S100. EMSA with recombinant protein. TRAN2B, TRA2-BETA, Htra2- IgM-based pre-mRNA extracts. specific beta, DKFZp686F18120. for in vitro splicing. activators of pre- mRNA splicing. Cell. 93(1): 139-148.

Unique 3’CDS (134609 – 134838)

GTGAGCACTGCAACAAAAAGGCTGTTTTCTCTCGGATCTCCAAATTTAAAAGCACAAGGAATGATTGTACCACACA AAGTAATGTAAAACATTAAAGGACTCATTAAAAAGTAACAGTTGTCTCATATCATCTTGATTTATTGTCACTGTTGC TAACTTTCAGGCTCGGAGGAGATGCTCCTCCCAAAATGAGTTCGGAGATGATAGCAGTAATAATGAGACCCCCGG GC

Art Protein Recognized icle Gene/Construct Binding Position Protein Notes PubMed ID Reference Splicing Assay Name Sequence not (Target RNA) Assay es

SELEX imposing a selection of the constructs for Smith PJ, Zhang splicing rather Gene Name and C, Wang J, Chew than for Synonymous: SL, Zhang MQ, Construct of binding. SFRS1, splicing Krainer AR. BRCA1 [672] EX17 Selection of the factor (2006) - INT17 - EX18 - constructs in arginine/serine-rich Functional An increased INT18 - EX19 with HeLa S100 1 (splicing factor 2, SELEX of specificity score random 7nt and extract alternate splicing random SF2/AS matrix for the 14nt inserted in complemented 167-173 CGGAGGA factor), ASF, SF2, 16825284 7nt and F prediction of EX18. Construct of by recombinant SF2p33, SRp30a, 14nt with SF2/ASF- SMN1 [6606] EX6 - protein. MGC5228. recombina specific exonic INT6 - EX7 - INT7 Winners are The shuttling nt protein. splicing - EX8 with 7nt confirmed by in protein SF2/ASF enhancers. SELEX-winners vitro splicing in binds TAP and can Hum Mol Genet. inserted in EX7. both HeLa function as export 15(16):2490- nuclear extract factors (18364396). 2508. and S100 extract complemented by recombinant protein.

Gene Name and Dye BT, Buvoli Contructs of UV Synonymous: M, Mayer SA, tropomyosin 1 alpha crosslink, SFRS1, splicing Lin CH, Patton TPM1 [7168] EX1 - In vitro splicing competitio factor JG. (1998) INT1 - EX2 - INT2 and competition n assay, arginine/serine-rich Enhancer - EX3 with WT and in HeLa cell Western 1 (splicing factor 2, elements activate mutants competitor nuclear extracts. blot, SF2/AS 168-174 GGAGGAG alternate splicing 9848651 the weak 3\' sequences of TPM1 In vivo splicing EMSA F factor), ASF, SF2, splice site of EX2 for in vitro into smooth using EX2 SF2p33, SRp30a, alpha- splicing. Construct muscle cells alpha-TM MGC5228. tropomyosin of TPM1 EX1 - (SMCs) and and The shuttling exon 2. INT1 - EX2 - INT2 HeLa cells. purified protein SF2/ASF RNA. 4(12): - EX3 - INT3 - EX4 proteins binds TAP and can 1523-1536. with wt or mutants or HeLa

145

function as export EX2 for in vivo nuclear factors (18364396). splicing. extracts.

Gene Name and Synonymo us: SFRS2, splicing factor Liu HX, Chew SL, arginine/ser Cartegni L, Zhang ine-rich 2, MQ, Krainer AR. SC-35, (2000) Constructs of mouse IgM SFRS2A, Exonic splicing EX_M1 - INT - EX_M2 S SRp30b, enhancer motif mutated by inserting 20nt 3-10 C GAGCACUG PR264. 10629063 recognized by randomized in EX_M2. 35 SC35 human SC35 under Construct of mouse IgM accelerates splicing EX_C3 - INT - EX_C4. transcriptio conditions. nal Mol Cell Biol. elongation 20(3): 1063-1071. (co- transcriptio nal splicing) (PMID: 18641664).

Gene Name and Synonymo us: SFRS2, In vitro splicing splicing factor and arginine/ser Dye BT, Buvoli Contructs of tropomyosin competitio ine-rich 2, M, Mayer SA, Lin 1 alpha TPM1 [7168] EX1 n in HeLa UV crosslink, SC-35, CH, Patton JG. - INT1 - EX2 - INT2 - cell competition SFRS2A, (1998) EX3 with WT and mutants nuclear assay, Western S SRp30b, Enhancer elements competitor sequences of extracts. blot, EMSA 168-174 C GGAGGAG PR264. 9848651 activate the weak TPM1 EX2 for in vitro In vivo using EX2 35 SC35 3\' splice site of splicing. Construct of splicing alpha-TM and accelerates alpha-tropomyosin TPM1 EX1 - INT1 - EX2 into purified proteins transcriptio exon 2. - INT2 - EX3 - INT3 - smooth or HeLa nuclear nal RNA. 4(12): 1523- EX4 with wt or mutants muscle extracts. elongation 1536. EX2 for in vivo splicing. cells (co- (SMCs) transcriptio and HeLa nal cells. splicing) (PMID: 18641664).

Gene Name and Synonymo us: SFRS2, splicing factor arginine/ser Rooke N, ine-rich 2, Markovtsov V, In vitro SC-35, Cagavi E, Black splicing UV crosslink SFRS2A, DL. (2003) with Weri- and S SRp30b, Roles for SR Construct of c-src [20779] 1, Weri-1 immunoprecipita 168-173 C GGAGGA PR264. 12612063 proteins and EX_N1. S100 and tion with Weri-1 35 SC35 hnRNP A1 in the HeLa and HeLa accelerates regulation of c-src nuclear nuclear extracts. transcriptio exon N1. extracts. nal Mol Cell Biol. elongation 23(6):1874-1884. (co- transcriptio nal splicing) (PMID: 18641664).

Gene Name Hallay H, Locker Competition S and N, Ayadi L, Sequences deriving from assays with 169-174 C GAGGAG 16990281 Synonymo Ropers D, Guittet HIV-1 Tat [155871] recombinant 35 us: SFRS2, E, Branlant C. protein

146

splicing (2006) factor Biochemical and arginine/ser NMR study on the ine-rich 2, competition SC-35, between proteins SFRS2A, SC35, SRp40, and SRp30b, heterogeneous PR264. nuclear SC35 ribonucleoprotein accelerates A1 at the HIV-1 transcriptio Tat exon 2 splicing nal site. elongation J Biol Chem. (co- 281(48):37159- transcriptio 37174. nal splicing) (PMID: 18641664).

Gene Name and Synonymo us: SFRS2, splicing factor arginine/ser ine-rich 2, Caputi M, Zahler SC-35, AM. (2002) SFRS2A, SR proteins and In vitro RNA affinity S SRp30b, hnRNP H regulate Construct of HIV-1 env splicing chromatography 170-174 C AGGAG PR264. 11847131 the splicing of the [155971] EX_6D and part with HeLa assay and 35 SC35 HIV-1 tev-specific of flanking introns. nuclear immunoblot. accelerates exon 6D. extracts transcriptio EMBO J. 21(4): nal 845-855. elongation (co- transcriptio nal splicing) (PMID: 18641664).

Gene Name and Synonymo us: SFRS2, splicing SELEX of 20-nt Cavaloc Y, factor random with Bourgeois CF, Sequences of 20 nt arginine/ser recombinant Kister L, Stevenin random for SELEX. ine-rich 2, protein. Winners J. (1999) Constructs of SC-35, confirmed by The splicing EXE1A_Adenovirus - SFRS2A, In vitro EMSA with factors 9G8 and partial_INT_E1A_Adenov S SRp30b, splicing in recombinant SRp20 irus - partial_INT_FN1 - 170-176 C AGGAGAU PR264. 10094314 HeLa protein, UV transactivate EX_ED1_FN1 of 35 SC35 S100 crosslink, splicing through FIBRONECTIN (FN1) accelerates extracts. complementatio different and [2335] for in vitro transcriptio n assay, specific splicing. Construct of Sp1 nal immunoprecipita enhancers. unit of Adenovirus E1A elongation tions with HeLa RNA. 5(3): 468- for in vitro splicing. (co- S100 and 483. transcriptio nuclear extracts. nal splicing) (PMID: 18641664).

Gene Name and Caputi M, Zahler Synonymo AM. (2002) us: SFRS2, SR proteins and In vitro RNA affinity S splicing hnRNP H regulate Construct of HIV-1 env splicing chromatography 206-210 C AGCAG factor 11847131 the splicing of the [155971] EX_6D and part with HeLa assay and 35 arginine/ser HIV-1 tev-specific of flanking introns. nuclear immunoblot. ine-rich 2, exon 6D. extracts SC-35, EMBO J. 21(4): SFRS2A, 845-855. SRp30b,

147

PR264. SC35 accelerates transcriptio nal elongation (co- transcriptio nal splicing) (PMID: 18641664).

Hargous Y, Hautbergue GM, Gene Name and Synonymous: Tintaru AM, Skrisovska L, SFRS3, splicing factor Golovanov AP, Stevenin J, Lian NMR arginine/serine-rich 3. 170360 LY, Wilson SA, Allain FH.(2006) Synthesized 35-38 SRp20 GAUC spectrosc The shuttling protein SRp20 44 Molecular basis of RNA sequences opy binds TAP and can function as recognition and TAP binding by export factors (18364396). the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Synonymous: Tintaru AM, Skrisovska L, SFRS3, splicing factor Golovanov AP, Stevenin J, Lian NMR UCAU arginine/serine-rich 3. 170360 LY, Wilson SA, Allain FH.(2006) Synthesized 128-132 SRp20 spectrosc C The shuttling protein SRp20 44 Molecular basis of RNA sequences opy binds TAP and can function as recognition and TAP binding by export factors (18364396). the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Synonymous: Tintaru AM, Skrisovska L, SFRS3, splicing factor Golovanov AP, Stevenin J, Lian NMR arginine/serine-rich 3. 170360 LY, Wilson SA, Allain FH.(2006) Synthesized 129-132 SRp20 CAUC spectrosc The shuttling protein SRp20 44 Molecular basis of RNA sequences opy binds TAP and can function as recognition and TAP binding by export factors (18364396). the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

SELEX imposing a selection of the constructs Liu HX, for splicing Zhang M, rather than Krainer AR. for binding. (1998) Construct of Selection of Identification EX_M1 - INT1 - the constructs of functional EX_M2 murine UV crosslink, Gene Name and by splicing in exonic splicing IgM and its competition and ACUG Synonymous: SFRS5, HeLa S100 7-11 SRp40 9649504 enhancer variants obtained immunoprecipitati C splicing factor extracts motifs by replacing the on assays in HeLa arginine/serine-rich 5, HRS. complemente recognized by natural ESE in the nuclear extracts. d by individual SR M2 exon with 20nt recombinant proteins. random. SR protein. Genes Dev. Winners are 12(13): 1998- confirmed by 2012. in vitro splicing in HeLa nuclear extracts.

SELEX Liu HX, imposing a Zhang M, selection of Krainer AR. the constructs (1998) Construct of for splicing Identification EX_M1 - INT1 - rather than of functional EX_M2 murine UV crosslink, Gene Name and for binding. exonic splicing IgM and its competition and AAAG Synonymous: SFRS5, Selection of 17-21 SRp40 9649504 enhancer variants obtained immunoprecipitati G splicing factor the constructs motifs by replacing the on assays in HeLa arginine/serine-rich 5, HRS. by splicing in recognized by natural ESE in the nuclear extracts. HeLa S100 individual SR M2 exon with 20nt extracts proteins. random. complemente Genes Dev. d by 12(13): 1998- recombinant 2012. SR protein.

148

Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for splicing Zhang M, rather than Krainer AR. for binding. (1998) Construct of Selection of Identification EX_M1 - INT1 - the constructs of functional EX_M2 murine UV crosslink, Gene Name and by splicing in exonic splicing IgM and its competition and AAAG Synonymous: SFRS5, HeLa S100 94-98 SRp40 9649504 enhancer variants obtained immunoprecipitati G splicing factor extracts motifs by replacing the on assays in HeLa arginine/serine-rich 5, HRS. complemente recognized by natural ESE in the nuclear extracts. d by individual SR M2 exon with 20nt recombinant proteins. random. SR protein. Genes Dev. Winners are 12(13): 1998- confirmed by 2012. in vitro splicing in HeLa nuclear extracts.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. AGCA SFRS9, splicing with HeLa 4-8 SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, C factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. AGCA SFRS9, splicing with HeLa 51-55 SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, C factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. AGGA SFRS9, splicing with HeLa 57-61 SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, A factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Gene Name and In vitro Cloutier P, Synthesized SELEX of 20nt random Synonymous: splicing Shkreta L, oligos. with recombinant protein. ACCA SFRS9, splicing with HeLa 69-73 SRp30c 17548433 Toutant J, Sequences of EMSA, UV crosslink, C factor nuclear Klarskov K, 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, Chabot B. (2007) for SELEX. nuclear extracts. 9. siRNA. hnRNP I/PTB can

149

antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. 96- AGGA SFRS9, splicing with HeLa SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, 100 C factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Antagonistic splicing Gene Name and effects of the Construct of assays in Synonymous: EMSA using recombinant SRp30c protein BCL2L1 HeLa 96- AGGA SFRS9, splicing protein. UV cross-linking SRp30c 18534987 and cryptic 5\' [600039] EX1- nuclear 100 C factor in HeLa and splice sites on the EX2-INT2- extracts arginine/serine-rich immunoprecipitation. alternative EX3 and 9. splicing of the recombina apoptotic nt protein regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. 167- CGGA SFRS9, splicing with HeLa SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, 171 G factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. 170- AGGA SFRS9, splicing with HeLa SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, 174 G factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta In vitro L, Goekjian S, splicing Gene Name and Revil T, Chabot Construct of assays in Synonymous: B. (2008) EMSA using recombinant BCL2L1 HeLa 170- AGGA SFRS9, splicing Antagonistic protein. UV cross-linking SRp30c 18534987 [600039] EX1- nuclear 174 G factor effects of the in HeLa and EX2-INT2- extracts arginine/serine-rich SRp30c protein immunoprecipitation. EX3 and 9. and cryptic 5\' recombina splice sites on the nt protein alternative splicing of the

150

apoptotic regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. 196- CGGA SFRS9, splicing with HeLa SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, 200 G factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro Klarskov K, Synthesized SELEX of 20nt random Synonymous: splicing Chabot B. (2007) oligos. with recombinant protein. 206- AGCA SFRS9, splicing with HeLa SRp30c 17548433 hnRNP I/PTB can Sequences of EMSA, UV crosslink, 210 G factor nuclear antagonize the 20nt random SDS-PAGE with HeLa arginine/serine-rich extracts, splicing repressor for SELEX. nuclear extracts. 9. siRNA. activity of SRp30c. RNA 13: 1287- 1300.

Exon 13, predominant 3’UTR ( 134839 – 138757) (results divided into searchable sections)

CCCAGCTCTGGGCCCCCCATTCAGGCCGAGGGGGCTGCTCCGGGGGGCCGACTTGGTGCACGTTTGGATTTGGAG GATCCCTGCACTGCCTTCTCTGTGTTTGTTGCTCTTGCTGTTTTCTCCTGCCTGATAAACAACAACTTGGGATGATCC TTTCCTTCCATTTTGATGCCAACCTCTTTTTATTTTTAAGTGTTGAAGCTGCACAAACTGAATAATTTAAACAAATGC TGGTTTCTGCCAAAGATGGACACGAATAAGTTAATTTTCCAGCTCAGAATGAGTACAGTTGAATTTGAGACTCTGT CGGACTTCTGCCTGGTTTTATTTGGGACTATTTCATCTGCTCTTGATTTGTAAATAGCACCTGGATAGCAAGTTATA ATGCTTATTTATTTGAAAATGCTTTTTTTTTTTTTACGTTAAGCACATTTATCTTGAACTGGAGCTTCTAAAATGGGC CCCAGGGGTGCAAGATGTTGGTGTAATTCAGAGATAGTAAAGGTTTATCGCAGTGTGAATTATAAGAGTCCATCC AAATCAACGTCCCCTCCCTCCTCTCATGCGATCCAGGTAATTATGCAGTTAGTGCCACAGTAGACTAGCCTAGCAAA GGGTTTGCTCCTTGCTGTCTCTGACTGCACCACACAGCTATTGATGGCAGCTGAAAGAAAGTGGATCATGCCTTAA TTTTAAATATTCCTGTCCTCTGGTTATTATTTTAAGGAACTTCATCATGTTAAAATGACAGCATTCAAAGGTGTACCA CAATCAATTTATCAAGGAAATAAAGGCTATTGTAACCAGAGATTTA

Splic Bindin Recognized Protein Article ing Position Protein Name PubMed ID Reference Gene/Construct (Target RNA) g Sequence Notes notes Assa Assay y

Gene Name Tacke R, In SELEX and Manley JL. vitro of Synonymou (1995) splic random s: SFRS1, The human ing 20nt Construct containing NCAM1 splicing splicing factors in with [4684] EX18, downstream 5' splice factor ASF/SF2 and HeL recomb 245-252 SF2/ASF AGAUGGAC 7543047 site, alpha globin HBA2 [3040] arginine/seri SC35 possess a inant INT2, HBA2 EX3. Sequences of 20 ne-rich 1 distinct, nucl protein. nt random for SELEX. (splicing functionally ear Confir factor 2, significant RNA and med by alternate binding S100 EMSA, splicing specificities. extra compet

151

factor), EMBO J. cts. ition ASF, SF2, 14(14): 3540- assay SF2p33, 3551. with SRp30a, recomb MGC5228. inant The protein shuttling and protein SELEX SF2/ASF winners binds TAP . UV- and can crosslin function as king export and factors immun (18364396). oprecip itation with Hela nuclear extract or S100 extract.

Gene Name and Synonymou s: SFRS1, splicing factor In arginine/seri vitro ne-rich 1 splic UV Rooke N, (splicing ing crosslin Markovtsov V, factor 2, with k and Cagavi E, Black alternate Weri immun DL. (2003) splicing -1, oprecip Roles for SR factor), Weri itation proteins and 758-763 SF2/ASF AAGGUG ASF, SF2, 12612063 Construct of c-src [20779] EX_N1. -1 with hnRNP A1 in SF2p33, S100 Weri-1 the regulation of SRp30a, and and c-src exon N1. MGC5228. HeL HeLa Mol Cell Biol. The a nuclear 23(6):1874- shuttling nucl extracts 1884. protein ear . SF2/ASF extra binds TAP cts. and can function as export factors (18364396).

Gene Name and Synony mous: SFRS2, splicing factor arginine/ serine- UV rich 2, crosslink SC-35, Rooke N, Markovtsov V, Cagavi and In vitro splicing SFRS2A E, Black DL. (2003) immunopr Construct of c- with Weri-1, , Roles for SR proteins and ecipitation 72-77 SC35 GGAGGA 12612063 src [20779] Weri-1 S100 and SRp30b, hnRNP A1 in the regulation of c- with EX_N1. HeLa nuclear PR264. src exon N1. Weri-1 extracts. SC35 Mol Cell Biol. 23(6):1874-1884. and HeLa accelerat nuclear es extracts. transcrip tional elongati on (co- transcrip tional splicing) (PMID: 1864166

152

4).

Gene Name and Synony mous: SFRS2, splicing factor Synthesized arginine/ oligos for UV- serine- crosslink. rich 2, Construct of UV SC-35, beta-globin Schaal TD, Maniatis T. (1999) crosslink SFRS2A [3043] EX1 - Multiple distinct splicing and SDS- , INT1 - EX2. In vitro splicing in 111- UGCUGU enhancers in the protein-coding PAGE SC35 SRp30b, 9858550 Construct and HeLa S100 117 U sequences of a constitutively with HeLa PR264. mutants of extracts. spliced pre-mRNA. S100 and SC35 beta-globin Mol Cell Biol. 19(1): 261-73. nuclear accelerat [3043] EX3 - extracts. es INT3 - transcrip partial_EX4 - tional EX2 for in elongati vitro splicing. on (co- transcrip tional splicing) (PMID: 1864166 4).

Gene Name and RNA Synony Affinity mous: Chromato SFRS2, graphy splicing Assays, factor SDS- arginine/ PAGE in serine- HeLa cell rich 2, Zahler AM, Damgaard CK, nuclear SC-35, Kjems J, Caputi M. (2004) extracts, SFRS2A SC35 and heterogeneous nuclear Nuclear , ribonucleoprotein A/B proteins Constructs of In Vitro Splicing 595- CAGUAG Extract SC35 SRp30b, 14703516 bind to a juxtaposed exonic HIV-1 Tat with HeLa S100 or 601 A Depletion PR264. splicing enhancer/exonic splicing [155871] EX2. nuclear extracts. by high SC35 silencer element to regulate HIV- affinity accelerat 1 tat exon 2 splicing. RNA. es J Biol Chem. 279(11): 10077-84. SDS- transcrip PAGE, tional immunobl elongati otting, on (co- RNA transcrip Footprinti tional ng splicing) Analysis. (PMID: 1864166 4).

Gene Name and Synony mous: SFRS2, Construct of RNA splicing Caputi M, Zahler AM. (2002) HIV-1 env affinity factor SR proteins and hnRNP H [155971] In vitro splicing chromatog 596- SC35 AGUAG arginine/ 11847131 regulate the splicing of the HIV- EX_6D and with HeLa nuclear raphy 600 serine- 1 tev-specific exon 6D. part of extracts assay and rich 2, EMBO J. 21(4): 845-855. flanking immunobl SC-35, introns. ot. SFRS2A , SRp30b, PR264. SC35

153

accelerat es transcrip tional elongati on (co- transcrip tional splicing) (PMID: 1864166 4).

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, The 170360 NMR 76-79 SRp20 GAUC Allain Synthesized sequences shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, 149- The 170360 NMR SRp20 GAUC Allain Synthesized sequences 152 shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

340- Gene Name 170360 Hargous NMR SRp20 UCAUC Synthesized sequences 344 and 44 Y, spectroscopy

154

Synonymou Hautberg s: SFRS3, ue GM, splicing Tintaru factor AM, arginine/ser Skrisovsk ine-rich 3. a L, The Golovano shuttling v AP, protein Stevenin SRp20 J, Lian binds TAP LY, and can Wilson function as SA, export Allain factors FH.(2006) (18364396). Molecular basis of RNA recognitio n and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, 341- The 170360 NMR SRp20 CAUC Allain Synthesized sequences 344 shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Gene Name Hargous and Y, Synonymou Hautberg s: SFRS3, ue GM, splicing Tintaru factor AM, arginine/ser Skrisovsk ine-rich 3. a L, 533- 170360 NMR SRp20 CAUC The Golovano Synthesized sequences 536 44 spectroscopy shuttling v AP, protein Stevenin SRp20 J, Lian binds TAP LY, and can Wilson function as SA, export Allain factors FH.(2006)

155

(18364396). Molecular basis of RNA recognitio n and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, 567- The 170360 NMR SRp20 GAUC Allain Synthesized sequences 570 shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, 678- The 170360 NMR SRp20 GAUC Allain Synthesized sequences 681 shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

156

Cavaloc Y, Bourgeois Gene Name CF, Kister and L, SELEX of 20-nt Synonymou Stevenin random with s: SFRS3, J. (1999) recombinant splicing The Sequences of 20 nt random for protein. Winners factor splicing SELEX. Constructs of confirmed by arginine/ser factors EXE1A_Adenovirus - EMSA with ine-rich 3. 9G8 and partial_INT_E1A_Adenovirus - In vitro recombinant 731- The 100943 SRp20 partial_INT_FN1 - splicing in SRp20 UUCAUCAU protein, UV 738 shuttling 14 transactiv EX_ED1_FN1 of HeLa S100 crosslink, protein ate FIBRONECTIN (FN1) [2335] extracts. complementation SRp20 splicing for in vitro splicing. Construct of assay, binds TAP through Sp1 unit of Adenovirus E1A for immunoprecipitati and can different in vitro splicing. ons with HeLa function as and S100 and nuclear export specific extracts. factors enhancers (18364396). . RNA. 5(3): 468- 483.

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, 732- The 170360 NMR SRp20 UCAUC Allain Synthesized sequences 736 shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Hargous Y, Gene Name Hautberg and ue GM, Synonymou Tintaru s: SFRS3, AM, splicing Skrisovsk factor a L, arginine/ser Golovano ine-rich 3. v AP, 733- The 170360 Stevenin NMR SRp20 CAUCAU Synthesized sequences 738 shuttling 44 J, Lian spectroscopy protein LY, SRp20 Wilson binds TAP SA, and can Allain function as FH.(2006) export Molecular factors basis of (18364396). RNA recognitio n and

157

TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

Hargous Y, Hautberg ue GM, Tintaru AM, Skrisovsk Gene Name a L, and Golovano Synonymou v AP, s: SFRS3, Stevenin splicing J, Lian factor LY, arginine/ser Wilson ine-rich 3. SA, 733- The 170360 NMR SRp20 CAUC Allain Synthesized sequences 736 shuttling 44 spectroscopy FH.(2006) protein Molecular SRp20 basis of binds TAP RNA and can recognitio function as n and export TAP factors binding (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):51 26-5137.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing enhancer and immunoprecipitation 7-11 SRp40 UCUGG SFRS5, 9649504 variants in HeLa motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

Gene Name Liu HX, Construct of SELEX UV crosslink, competition 34- SRp40 GCUGC and 9649504 Zhang M, EX_M1 - imposing and immunoprecipitation 38 Synonymous: Krainer INT1 - a assays in HeLa nuclear

158

SFRS5, AR. EX_M2 selection extracts. splicing factor (1998) murine IgM of the arginine/serine Identifica and its constructs -rich 5, HRS. tion of variants for functiona obtained by splicing l exonic replacing the rather splicing natural ESE in than for enhancer the M2 exon binding. motifs with 20nt Selection recognize random. of the d by constructs individua by l SR splicing proteins. in HeLa Genes S100 Dev. extracts 12(13): complem 1998- ented by 2012. recombin ant SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 85- enhancer and immunoprecipitation SRp40 ACUGC SFRS5, 9649504 variants in HeLa 89 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

Liu HX, SELEX Construct of Zhang M, imposing EX_M1 - Krainer a INT1 - AR. selection Gene Name EX_M2 (1998) of the and murine IgM Identifica constructs UV crosslink, competition Synonymous: and its 201- tion of for and immunoprecipitation SRp40 GCUGC SFRS5, 9649504 variants 205 functiona splicing assays in HeLa nuclear splicing factor obtained by l exonic rather extracts. arginine/serine replacing the splicing than for -rich 5, HRS. natural ESE in enhancer binding. the M2 exon motifs Selection with 20nt recognize of the random. d by constructs

159

individua by l SR splicing proteins. in HeLa Genes S100 Dev. extracts 12(13): complem 1998- ented by 2012. recombin ant SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 251- enhancer and immunoprecipitation SRp40 ACACG SFRS5, 9649504 variants in HeLa 255 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

SELEX imposing Liu HX, a Zhang M, selection Krainer of the AR. constructs (1998) Construct of for Identifica EX_M1 - splicing tion of INT1 - rather functiona Gene Name EX_M2 than for l exonic and murine IgM binding. splicing UV crosslink, competition Synonymous: and its Selection 442- enhancer and immunoprecipitation SRp40 ACUGG SFRS5, 9649504 variants of the 446 motifs assays in HeLa nuclear splicing factor obtained by constructs recognize extracts. arginine/serine replacing the by d by -rich 5, HRS. natural ESE in splicing individua the M2 exon in HeLa l SR with 20nt S100 proteins. random. extracts Genes complem Dev. ented by 12(13): recombin 1998- ant SR 2012. protein. Winners

160

are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 501- enhancer and immunoprecipitation SRp40 AAAGG SFRS5, 9649504 variants in HeLa 505 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 612- enhancer and immunoprecipitation SRp40 AAAGG SFRS5, 9649504 variants in HeLa 616 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

638- Gene Name Liu HX, Construct of SELEX UV crosslink, competition SRp40 ACUGC 9649504 642 and Zhang M, EX_M1 - imposing and immunoprecipitation

161

Synonymous: Krainer INT1 - a assays in HeLa nuclear SFRS5, AR. EX_M2 selection extracts. splicing factor (1998) murine IgM of the arginine/serine Identifica and its constructs -rich 5, HRS. tion of variants for functiona obtained by splicing l exonic replacing the rather splicing natural ESE in than for enhancer the M2 exon binding. motifs with 20nt Selection recognize random. of the d by constructs individua by l SR splicing proteins. in HeLa Genes S100 Dev. extracts 12(13): complem 1998- ented by 2012. recombin ant SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 648- enhancer and immunoprecipitation SRp40 ACAGC SFRS5, 9649504 variants in HeLa 652 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

Liu HX, Construct of SELEX Zhang M, EX_M1 - imposing Krainer INT1 - a Gene Name AR. EX_M2 selection and (1998) murine IgM of the UV crosslink, competition Synonymous: Identifica and its constructs 661- and immunoprecipitation SRp40 GCAGC SFRS5, 9649504 tion of variants for 665 assays in HeLa nuclear splicing factor functiona obtained by splicing extracts. arginine/serine l exonic replacing the rather -rich 5, HRS. splicing natural ESE in than for enhancer the M2 exon binding. motifs with 20nt Selection recognize random. of the

162

d by constructs individua by l SR splicing proteins. in HeLa Genes S100 Dev. extracts 12(13): complem 1998- ented by 2012. recombin ant SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 709- enhancer and immunoprecipitation SRp40 UCUGG SFRS5, 9649504 variants in HeLa 713 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

SELEX Liu HX, imposing Zhang M, a Krainer selection AR. of the (1998) constructs Construct of Identifica for EX_M1 - tion of splicing INT1 - functiona rather Gene Name EX_M2 l exonic than for and murine IgM splicing binding. UV crosslink, competition Synonymous: and its 748- enhancer Selection and immunoprecipitation SRp40 ACAGC SFRS5, 9649504 variants 752 motifs of the assays in HeLa nuclear splicing factor obtained by recognize constructs extracts. arginine/serine replacing the d by by -rich 5, HRS. natural ESE in individua splicing the M2 exon l SR in HeLa with 20nt proteins. S100 random. Genes extracts Dev. complem 12(13): ented by 1998- recombin 2012. ant SR protein.

163

Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the constructs Liu HX, for Zhang M, splicing Krainer rather AR. than for (1998) Construct of binding. Identifica EX_M1 - Selection tion of INT1 - of the functiona Gene Name EX_M2 constructs l exonic and murine IgM by splicing UV crosslink, competition Synonymous: and its splicing 757- enhancer and immunoprecipitation SRp40 AAAGG SFRS5, 9649504 variants in HeLa 761 motifs assays in HeLa nuclear splicing factor obtained by S100 recognize extracts. arginine/serine replacing the extracts d by -rich 5, HRS. natural ESE in complem individua the M2 exon ented by l SR with 20nt recombin proteins. random. ant SR Genes protein. Dev. Winners 12(13): are 1998- confirmed 2012. by in vitro splicing in HeLa nuclear extracts.

Chandrad as S, Deikus G, Tardos JG, Bogdano v VY. In this (2010) context, Antagoni SRp40 and stic roles SC35 of four Gene Name antagonize SR Construct of and other SR proteins F3 [2152] In vivo Synonymous: proteins by Mutagenesis, splicing 788- in the EX4-INT4- splicing SRp40 AUAAAGG SFRS5, 19843576 competing assays, EMSA, 794 biosynthe EX5-INT5- in THP-1 splicing factor for certain Immunoblot. sis of EX6 and cells. arginine/serine sites in exon alternativ mutants -rich 5, HRS. 5, thereby ely promoting spliced TF (tissue tissue factor) exon factor 5 exclusion. transcript s in monocyti c cells. J Leukoc Biol. 87(1):147 -152.

Gene Name Liu HX, Construct of SELEX UV crosslink, competition 790- and Zhang M, EX_M1 - imposing and immunoprecipitation SRp40 AAAGG 9649504 794 Synonymous: Krainer INT1 - a assays in HeLa nuclear SFRS5, AR. EX_M2 selection extracts.

164

splicing factor (1998) murine IgM of the arginine/serine Identifica and its constructs -rich 5, HRS. tion of variants for functiona obtained by splicing l exonic replacing the rather splicing natural ESE in than for enhancer the M2 exon binding. motifs with 20nt Selection recognize random. of the d by constructs individua by l SR splicing proteins. in HeLa Genes S100 Dev. extracts 12(13): complem 1998- ented by 2012. recombin ant SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with Synonymous: K, Chabot B. (2007) 65 Synthesized oligos. splicing with recombinant SFRS9, splicing 1754843 hnRNP I/PTB can - SRp30c UGGAU Sequences of 20nt HeLa nuclear protein. EMSA, UV factor 3 antagonize the 69 random for SELEX. extracts, crosslink, SDS- arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with Synonymous: K, Chabot B. (2007) 74 Synthesized oligos. splicing with recombinant SFRS9, splicing 1754843 hnRNP I/PTB can - SRp30c AGGAU Sequences of 20nt HeLa nuclear protein. EMSA, UV factor 3 antagonize the 78 random for SELEX. extracts, crosslink, SDS- arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 24 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 8- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c UGGAC Sequences of 20nt HeLa nuclear protein. EMSA, UV 25 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 2 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 30 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 8- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c CGGAC Sequences of 20nt HeLa nuclear protein. EMSA, UV 31 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 2 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Gene Name and Paradis C, Cloutier In vitro SELEX of 20nt 36 Synonymous: P, Shkreta L, Synthesized oligos. splicing with random with 3- 1754843 SRp30c AGCAC SFRS9, splicing Toutant J, Klarskov Sequences of 20nt HeLa nuclear recombinant 36 3 factor K, Chabot B. (2007) random for SELEX. extracts, protein. EMSA, UV 7 arginine/serine- hnRNP I/PTB can siRNA. crosslink, SDS-

165

rich 9. antagonize the PAGE with HeLa splicing repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 36 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 9- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c UGGAU Sequences of 20nt HeLa nuclear protein. EMSA, UV 37 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 3 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 42 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 6- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c AGCAC Sequences of 20nt HeLa nuclear protein. EMSA, UV 43 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 0 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 64 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 3- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c ACCAC Sequences of 20nt HeLa nuclear protein. EMSA, UV 64 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 7 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 67 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 6- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c UGGAU Sequences of 20nt HeLa nuclear protein. EMSA, UV 68 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 0 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 72 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 5- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c AGGAA Sequences of 20nt HeLa nuclear protein. EMSA, UV 72 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 9 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, SELEX of 20nt Gene Name and Toutant J, Klarskov In vitro random with 76 Synonymous: K, Chabot B. (2007) Synthesized oligos. splicing with recombinant 5- SFRS9, splicing 1754843 hnRNP I/PTB can SRp30c ACCAC Sequences of 20nt HeLa nuclear protein. EMSA, UV 76 factor 3 antagonize the random for SELEX. extracts, crosslink, SDS- 9 arginine/serine- splicing repressor siRNA. PAGE with HeLa rich 9. activity of SRp30c. nuclear extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier SELEX of 20nt Gene Name and P, Shkreta L, In vitro random with 78 Synonymous: Toutant J, Klarskov Synthesized oligos. splicing with recombinant 3- SFRS9, splicing 1754843 K, Chabot B. (2007) SRp30c AGGAA Sequences of 20nt HeLa nuclear protein. EMSA, UV 78 factor 3 hnRNP I/PTB can random for SELEX. extracts, crosslink, SDS- 7 arginine/serine- antagonize the siRNA. PAGE with HeLa rich 9. splicing repressor nuclear extracts. activity of SRp30c.

166

RNA 13: 1287- 1300.

Gene Name and Synonymous: Ray D, Kazan H, FUSIP1, FUS interacting protein Chan ET, Pena (serine/arginine-rich) 1, NSSR, Castillo L, Chaudhry TASR, SRp38, TASR1, TASR2, S, Talukder S, FUSIP2, SFRS13, SRrp40. Blencowe BJ, Morris 66 Dephosphorylation converts SRp38 to Q, Hughes TR. RNAcompete 7- a splicing repressor (PMID: (2009) Synthesized using SRp38 GAAAGAA 19561594 67 12419250) Rapid and systematic sequences recombinant 3 SRp38 is an atypical SR protein that analysis of the RNA protein functions as a general splicing recognition repressor when dephosphorylated, but specificities of RNA- when phosphorylated it functions as a binding proteins. sequence-specific splicing activator Nat Biotechnol. (PMID: 18794844). 27(7):667-670.

Gene Name and Synonymous: Ray D, Kazan H, FUSIP1, FUS interacting protein Chan ET, Pena (serine/arginine-rich) 1, NSSR, Castillo L, Chaudhry TASR, SRp38, TASR1, TASR2, S, Talukder S, FUSIP2, SFRS13, SRrp40. Blencowe BJ, Morris 66 Dephosphorylation converts SRp38 to Q, Hughes TR. RNAcompete 8- a splicing repressor (PMID: (2009) Synthesized using SRp38 AAAGAAA 19561594 67 12419250) Rapid and systematic sequences recombinant 4 SRp38 is an atypical SR protein that analysis of the RNA protein functions as a general splicing recognition repressor when dephosphorylated, but specificities of RNA- when phosphorylated it functions as a binding proteins. sequence-specific splicing activator Nat Biotechnol. (PMID: 18794844). 27(7):667-670.

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 669- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 673 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Watanabe S, Gene Name and Terada T, Synonymous: SFRS10, Kobayashi N, splicing factor Shirouzu M, arginine/serine-rich 10 Kigawa T, Tanaka 541- Synthesized NMR HTra2beta1 UCAAC (transformer 2 homolog, 20926394 A, Sugano S, 545 sequences spectroscopy Drosophila), TRA2B, GÃ¼ntert P, SRFS10, TRAN2B, Yokoyama S, TRA2-BETA, Htra2-beta, Muto Y.(2010) DKFZp686F18120. Structural basis for the dual RNA- recognition modes of human Tra2-Î² RRM. Nucleic Acids Res. 39(4):1538-1553.

Sequences of 20 Tacke R, Tohyama SELEX of 20nt Gene Name and nt random for M, Ogawa S, random with Synonymous: SFRS10, SELEX. Manley JL. (1998) In vitro recombinant splicing factor Sequence of Human Tra2 splicing protein, arginine/serine-rich 10 beta-globin 669- proteins are in HeLa confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 [3043] and 673 sequence-specific S100 and EMSA in HeLa Drosophila), TRA2B, constructs of activators of pre- nuclear nuclear extract SRFS10, TRAN2B, murine IgM- mRNA splicing. extracts. and S100. EMSA TRA2-BETA, Htra2-beta, based pre- Cell. 93(1): 139- with recombinant DKFZp686F18120. mRNA for in 148. protein. vitro splicing.

167

ATGCATTCTTCTAAATGTAAATTTAAAATTTGCCCTTTAAAAAAGTCCACTTTCCCCATATGCAAATGTTAATAGGAT TTTTATGGGGATTAAGAAGCGGCAAAACTACAGAAGCAGAATTCAAAGTAATTAAAAAAATACACACCAGTTTTA AATCAAGAGAAGTTGTAATCTCTTGTTTTAAGCTTGCGTTTGAGGGAAAATGACTTTTTCACCAATTTAGTATGCAT TGTTCTGTTGTTTTTATTTATGATTGATCATTATATGTGACTTGCATAAACTATTTAAAAAAAAAAACTATAATGACC AAAATAGCCATGGCTGAGAAACACAGTGGCTGGGCAGTTCAATAGGAGGTGACAATATGACAACTTCTCAAGCTT GGGAACTCACCAGACTGTTTCCTCCTTTAGGTAACAGATTCTGTCCCACGGCTAAACTTGTCTTTCACGTGGGAATT GCTTTTGTCAAACGTGAAAGAGTAAACAATAGCATTTCCCCAGAATGCCAGTTTTATGGAGCCCCAAATGCTCTGA AAACAATTAGTAACCTGGAAGTTGTCAGCCCAAAGGAAAGAAAAATCAATTGTATCTTGAAATTTTACCTATGGCT CTTTGGCCTGGCTTCTTTGTTCATTATAAGTTAGTGTGTTCCTTCAGGAAACAATGCCTTAATACCATAGAACATGG GGGCCTTAATAGTTGCTAACATTAAAAAAGCAAACAGAATGATTGAGGGATCCTTATGAAAACAAAATGGTGAAT TGGACATGCAGAACCTACCATTTCCTTCCCCTGTTTGCAATTTTTGT

Artic Bindi Protein Recognized Protein Splicing Position PubMed ID Reference le Gene/Construct (Target RNA) ng Name Sequence Notes Assay notes Assay

SELEX imposing Gene a Name and selection Synonymo of the us: SFRS1, constructs splicing for factor splicing arginine/se UV rather rine-rich 1 crossli than for (splicing Liu HX, Zhang nk, binding. factor 2, M, Krainer AR. comp Selection alternate (1998) etition of the splicing Identification and constructs factor), of functional Construct of EX_M1 - INT1 - immu by ASF, SF2, exonic splicing EX_M2 murine IgM and its nopre splicing 330-336 SF2/ASF CACAGUG SF2p33, 9649504 enhancer motifs variants obtained by replacing the cipitat in HeLa SRp30a, recognized by natural ESE in the M2 exon with ion S100 MGC5228 individual SR 20nt random. assays extracts . proteins. in compleme The Genes Dev. HeLa nted by shuttling 12(13): 1998- nuclea recombin protein 2012. r ant SR SF2/ASF extrac protein. binds TAP ts. Winners and can are function as confirmed export by in vitro factors splicing (18364396 in HeLa ). nuclear extracts.

Gene Name and Synonym Caputi M, Zahler ous: AM. (2002) RNA SFRS2, SR proteins and affinity splicing Construct of HIV-1 hnRNP H regulate In vitro splicing chromato factor env [155971] 93-97 SC35 AGAAG 11847131 the splicing of the with HeLa graphy arginine/s EX_6D and part of HIV-1 tev-specific nuclear extracts assay and erine-rich flanking introns. exon 6D. immunobl 2, SC-35, EMBO J. 21(4): 845- ot. SFRS2A, 855. SRp30b, PR264. SC35

168

accelerate s transcripti onal elongation (co- transcripti onal splicing) (PMID: 18641664 ).

Gene Name and Synonym ous: SFRS2, splicing factor arginine/s erine-rich Caputi M, Zahler 2, SC-35, AM. (2002) RNA SFRS2A, SR proteins and affinity SRp30b, Construct of HIV-1 hnRNP H regulate In vitro splicing chromato PR264. env [155971] 110-114 SC35 AGAAG 11847131 the splicing of the with HeLa graphy SC35 EX_6D and part of HIV-1 tev-specific nuclear extracts assay and accelerate flanking introns. exon 6D. immunobl s EMBO J. 21(4): 845- ot. transcripti 855. onal elongation (co- transcripti onal splicing) (PMID: 18641664 ).

Gene Name and Synonym ous: SFRS2, splicing factor arginine/s erine-rich Caputi M, Zahler 2, SC-35, AM. (2002) RNA SFRS2A, SR proteins and affinity SRp30b, Construct of HIV-1 hnRNP H regulate In vitro splicing chromato PR264. env [155971] 113-117 SC35 AGCAG 11847131 the splicing of the with HeLa graphy SC35 EX_6D and part of HIV-1 tev-specific nuclear extracts assay and accelerate flanking introns. exon 6D. immunobl s EMBO J. 21(4): 845- ot. transcripti 855. onal elongation (co- transcripti onal splicing) (PMID: 18641664 ).

Gene Name and Caputi M, Zahler Synonym AM. (2002) RNA ous: SR proteins and affinity SFRS2, Construct of HIV-1 hnRNP H regulate In vitro splicing chromato splicing env [155971] 161-165 SC35 AGAAG 11847131 the splicing of the with HeLa graphy factor EX_6D and part of HIV-1 tev-specific nuclear extracts assay and arginine/s flanking introns. exon 6D. immunobl erine-rich EMBO J. 21(4): 845- ot. 2, SC-35, 855. SFRS2A, SRp30b,

169

PR264. SC35 accelerate s transcripti onal elongation (co- transcripti onal splicing) (PMID: 18641664 ).

Gene Name and Synonym ous: SFRS2, splicing factor arginine/s erine-rich Caputi M, Zahler 2, SC-35, AM. (2002) RNA SFRS2A, SR proteins and affinity SRp30b, Construct of HIV-1 hnRNP H regulate In vitro splicing chromato PR264. env [155971] 352-356 SC35 AGGAG 11847131 the splicing of the with HeLa graphy SC35 EX_6D and part of HIV-1 tev-specific nuclear extracts assay and accelerate flanking introns. exon 6D. immunobl s EMBO J. 21(4): 845- ot. transcripti 855. onal elongation (co- transcripti onal splicing) (PMID: 18641664 ).

Gene Name and Hargous Y, Hautbergue GM, Tintaru Synonymous: SFRS3, AM, Skrisovska L, Golovanov AP, splicing factor Stevenin J, Lian LY, Wilson SA, NMR arginine/serine-rich 3. 170360 Allain FH.(2006) Synthesized 211-215 SRp20 UUCAC spectro The shuttling protein 44 Molecular basis of RNA recognition sequences scopy SRp20 binds TAP and and TAP binding by the SR proteins can function as export SRp20 and 9G8. factors (18364396). EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, Tintaru Synonymous: SFRS3, AM, Skrisovska L, Golovanov AP, splicing factor Stevenin J, Lian LY, Wilson SA, NMR arginine/serine-rich 3. 170360 Allain FH.(2006) Synthesized 256-259 SRp20 GAUC spectro The shuttling protein 44 Molecular basis of RNA recognition sequences scopy SRp20 binds TAP and and TAP binding by the SR proteins can function as export SRp20 and 9G8. factors (18364396). EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, Tintaru Synonymous: SFRS3, AM, Skrisovska L, Golovanov AP, splicing factor Stevenin J, Lian LY, Wilson SA, NMR arginine/serine-rich 3. 170360 Allain FH.(2006) Synthesized 447-451 SRp20 UUCAC spectro The shuttling protein 44 Molecular basis of RNA recognition sequences scopy SRp20 binds TAP and and TAP binding by the SR proteins can function as export SRp20 and 9G8. factors (18364396). EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, Tintaru Synonymous: SFRS3, AM, Skrisovska L, Golovanov AP, splicing factor Stevenin J, Lian LY, Wilson SA, NMR arginine/serine-rich 3. 170360 Allain FH.(2006) Synthesized 738-741 SRp20 GAUC spectro The shuttling protein 44 Molecular basis of RNA recognition sequences scopy SRp20 binds TAP and and TAP binding by the SR proteins can function as export SRp20 and 9G8. factors (18364396). EMBO J. 25(21):5126-5137.

142-146 SRp40 ACACC Gene 9649504 Liu HX, Zhang Construct of SELEX UV crosslink, competition

170

Name M, Krainer AR. EX_M1 - imposing a and immunoprecipitation and (1998) INT1 - selection of the assays in HeLa nuclear Synony Identification EX_M2 constructs for extracts. mous: of functional murine IgM splicing rather SFRS5, exonic splicing and its than for binding. splicing enhancer motifs variants Selection of the factor recognized by obtained by constructs by arginine individual SR replacing the splicing in HeLa /serine- proteins. natural ESE S100 extracts rich 5, Genes Dev. in the M2 complemented HRS. 12(13): 1998- exon with by recombinant 2012. 20nt random. SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the Liu HX, Zhang Construct of Gene constructs for M, Krainer AR. EX_M1 - Name splicing rather (1998) INT1 - and than for binding. Identification EX_M2 Synony Selection of the of functional murine IgM mous: constructs by UV crosslink, competition exonic splicing and its SFRS5, splicing in HeLa and immunoprecipitation 431-435 SRp40 ACGGC 9649504 enhancer motifs variants splicing S100 extracts assays in HeLa nuclear recognized by obtained by factor complemented extracts. individual SR replacing the arginine by recombinant proteins. natural ESE /serine- SR protein. Genes Dev. in the M2 rich 5, Winners are 12(13): 1998- exon with HRS. confirmed by in 2012. 20nt random. vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of the Liu HX, Zhang Construct of Gene constructs for M, Krainer AR. EX_M1 - Name splicing rather (1998) INT1 - and than for binding. Identification EX_M2 Synony Selection of the of functional murine IgM mous: constructs by UV crosslink, competition exonic splicing and its SFRS5, splicing in HeLa and immunoprecipitation 568-572 SRp40 AAAGG 9649504 enhancer motifs variants splicing S100 extracts assays in HeLa nuclear recognized by obtained by factor complemented extracts. individual SR replacing the arginine by recombinant proteins. natural ESE /serine- SR protein. Genes Dev. in the M2 rich 5, Winners are 12(13): 1998- exon with HRS. confirmed by in 2012. 20nt random. vitro splicing in HeLa nuclear extracts.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name Klarskov K, SELEX of 20nt random and Chabot B. Synthesized oligos. In vitro splicing with recombinant Synonymous: (2007) 1754843 Sequences of 20nt with HeLa protein. EMSA, UV 74-78 SRp30c AGGAU SFRS9, hnRNP I/PTB 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor can antagonize SELEX. siRNA. with HeLa nuclear arginine/serine the splicing extracts. -rich 9. repressor activity of SRp30c. RNA 13: 1287- 1300.

Gene Name Paradis C, SELEX of 20nt random and Cloutier P, Synthesized oligos. In vitro splicing with recombinant Synonymous: Shkreta L, 113- 1754843 Sequences of 20nt with HeLa protein. EMSA, UV SRp30c AGCAG SFRS9, Toutant J, 117 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor Klarskov K, SELEX. siRNA. with HeLa nuclear arginine/serine Chabot B. extracts. -rich 9. (2007)

171

hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name Klarskov K, SELEX of 20nt random and Chabot B. Synthesized oligos. In vitro splicing with recombinant Synonymous: (2007) 352- 1754843 Sequences of 20nt with HeLa protein. EMSA, UV SRp30c AGGAG SFRS9, hnRNP I/PTB 356 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor can antagonize SELEX. siRNA. with HeLa nuclear arginine/serine the splicing extracts. -rich 9. repressor activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) Gene Name Antagonistic and effects of the In vitro splicing EMSA using Construct of Synonymous: SRp30c protein assays in HeLa recombinant protein. UV 352- 1853498 BCL2L1 [600039] SRp30c AGGAG SFRS9, and cryptic 5\' nuclear extracts cross-linking in HeLa 356 7 EX1-EX2-INT2- splicing factor splice sites on and recombinant and EX3 arginine/serine the alternative protein immunoprecipitation. -rich 9. splicing of the apoptotic regulator Bcl- x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name Klarskov K, SELEX of 20nt random and Chabot B. Synthesized oligos. In vitro splicing with recombinant Synonymous: (2007) 570- 1754843 Sequences of 20nt with HeLa protein. EMSA, UV SRp30c AGGAA SFRS9, hnRNP I/PTB 574 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor can antagonize SELEX. siRNA. with HeLa nuclear arginine/serine the splicing extracts. -rich 9. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name Klarskov K, SELEX of 20nt random and Chabot B. Synthesized oligos. In vitro splicing with recombinant Synonymous: (2007) 658- 1754843 Sequences of 20nt with HeLa protein. EMSA, UV SRp30c AGGAA SFRS9, hnRNP I/PTB 662 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor can antagonize SELEX. siRNA. with HeLa nuclear arginine/serine the splicing extracts. -rich 9. repressor activity of SRp30c. RNA 13: 1287- 1300.

681- Gene Name 1754843 Paradis C, Synthesized oligos. In vitro splicing SELEX of 20nt random SRp30c AGAAC 685 and 3 Cloutier P, Sequences of 20nt with HeLa with recombinant

172

Synonymous: Shkreta L, random for nuclear extracts, protein. EMSA, UV SFRS9, Toutant J, SELEX. siRNA. crosslink, SDS-PAGE splicing factor Klarskov K, with HeLa nuclear arginine/serine Chabot B. extracts. -rich 9. (2007) hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name Klarskov K, SELEX of 20nt random and Chabot B. Synthesized oligos. In vitro splicing with recombinant Synonymous: (2007) 765- 1754843 Sequences of 20nt with HeLa protein. EMSA, UV SRp30c UGGAC SFRS9, hnRNP I/PTB 769 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor can antagonize SELEX. siRNA. with HeLa nuclear arginine/serine the splicing extracts. -rich 9. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name Klarskov K, SELEX of 20nt random and Chabot B. Synthesized oligos. In vitro splicing with recombinant Synonymous: (2007) 774- 1754843 Sequences of 20nt with HeLa protein. EMSA, UV SRp30c AGAAC SFRS9, hnRNP I/PTB 778 3 random for nuclear extracts, crosslink, SDS-PAGE splicing factor can antagonize SELEX. siRNA. with HeLa nuclear arginine/serine the splicing extracts. -rich 9. repressor activity of SRp30c. RNA 13: 1287- 1300.

Ray D, Kazan H, Chan ET, Gene Name and Synonymous: Pena Castillo L, FUSIP1, FUS interacting protein Chaudhry S, (serine/arginine-rich) 1, NSSR, Talukder S, TASR, SRp38, TASR1, TASR2, Blencowe BJ, FUSIP2, SFRS13, SRrp40. Morris Q, Dephosphorylation converts Hughes TR. 72 RNAcompete SRp38 to a splicing repressor (2009) - Synthesized using SRp38 GAAAGAA (PMID: 12419250) 19561594 Rapid and 57 sequences recombinant SRp38 is an atypical SR protein systematic 8 protein that functions as a general splicing analysis of the repressor when dephosphorylated, RNA but when phosphorylated it recognition functions as a sequence-specific specificities of splicing activator (PMID: RNA-binding 18794844). proteins. Nat Biotechnol. 27(7):667-670.

Gene Name and Synonymous: Ray D, Kazan FUSIP1, FUS interacting protein H, Chan ET, (serine/arginine-rich) 1, NSSR, Pena Castillo L, TASR, SRp38, TASR1, TASR2, Chaudhry S, FUSIP2, SFRS13, SRrp40. Talukder S, 57 RNAcompete Dephosphorylation converts Blencowe BJ, 3- Synthesized using SRp38 AAAGAAA SRp38 to a splicing repressor 19561594 Morris Q, 57 sequences recombinant (PMID: 12419250) Hughes TR. 9 protein SRp38 is an atypical SR protein (2009) that functions as a general splicing Rapid and repressor when dephosphorylated, systematic but when phosphorylated it analysis of the functions as a sequence-specific RNA

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splicing activator (PMID: recognition 18794844). specificities of RNA-binding proteins. Nat Biotechnol. 27(7):667-670.

Wu JY, Kar A, Kuo D, Yu B, Havlioglu N. (2006) Construct of Tau Positive clones identified Gene Name and SRp54 (SFRS11), a MAPT [4137] by fluorescence-activated Synonymous: regulator for tau exon 10 EX9 - INT9 - In vivo cell sorting and visual 92- SFRS11, splicing SRp54 AAGAAG 16943417 alternative splicing EX10 - INT10 - splicing in inspection. Confirmed by 97 factor, identified by an EX11 with GFP HEK293. UV crosslink, arginine/serine-rich expression cloning cDNA inserted immunoprecipitation, 11, p54. strategy. into EX11. SDS-PAGE. Mol Cell Biol. 26(18):6739-6747.

Tacke R, Tohyama M, Ogawa S, Manley JL. Sequences of 20 nt (1998) random for SELEX. SELEX of 20nt random In vitro Gene Name and Human Tra2 Sequence of beta- with recombinant protein, splicing in 92- Synonymous: proteins are globin [3043] and confirmed by EMSA in HTra2alpha AAGAA 9546399 HeLa S100 96 TRA2A, transformer- sequence- constructs of murine HeLa nuclear extract and and nuclear 2 alpha, HSU53209. specific IgM-based pre- S100. EMSA with extracts. activators of mRNA for in vitro recombinant protein. pre-mRNA splicing. splicing. Cell. 93(1): 139-148.

Tacke R, Tohyama M, Ogawa S, Manley JL. Sequences of 20 nt (1998) random for SELEX. SELEX of 20nt random In vitro Gene Name and Human Tra2 Sequence of beta- with recombinant protein, splicing in 574- Synonymous: proteins are globin [3043] and confirmed by EMSA in HTra2alpha AAGAA 9546399 HeLa S100 578 TRA2A, transformer- sequence- constructs of murine HeLa nuclear extract and and nuclear 2 alpha, HSU53209. specific IgM-based pre- S100. EMSA with extracts. activators of mRNA for in vitro recombinant protein. pre-mRNA splicing. splicing. Cell. 93(1): 139-148.

Tacke R, Sequences Gene Name and Tohyama M, of 20 nt Synonymous: Ogawa S, random for SFRS10, splicing Manley JL. SELEX. SELEX of 20nt factor (1998) Sequence In vitro random with arginine/serine-rich Human Tra2 of beta- splicing in recombinant protein, 10 (transformer 2 proteins are globin HeLa confirmed by EMSA 2-96 HTra2beta1 AAGAA 9546399 homolog, sequence- [3043] and S100 and in HeLa nuclear Drosophila), TRA2B, specific constructs nuclear extract and S100. SRFS10, TRAN2B, activators of of murine extracts. EMSA with TRA2-BETA, Htra2- pre-mRNA IgM-based recombinant protein. beta, splicing. pre-mRNA DKFZp686F18120. Cell. 93(1): for in vitro 139-148. splicing.

Wu JY, Kar A, Kuo D, Yu Gene Name and B, Havlioglu Construct Synonymous: N. (2006) of Tau SFRS10, splicing Positive clones SRp54 MAPT factor identified by (SFRS11), a [4137] EX9 arginine/serine-rich fluorescence-activated regulator for - INT9 - In vivo 92- 10 (transformer 2 cell sorting and visual HTra2beta1 AAGAAG 16943417 tau exon 10 EX10 - splicing in 97 homolog, inspection. Confirmed alternative INT10 - HEK293. Drosophila), TRA2B, by UV crosslink, splicing EX11 with SRFS10, TRAN2B, immunoprecipitation, identified by GFP cDNA TRA2-BETA, Htra2- SDS-PAGE. an expression inserted beta, cloning into EX11. DKFZp686F18120. strategy. Mol Cell

174

Biol. 26(18):6739- 6747.

Tacke R, Sequences Gene Name and Tohyama M, of 20 nt Synonymous: Ogawa S, random for SFRS10, splicing Manley JL. SELEX. SELEX of 20nt factor (1998) Sequence In vitro random with arginine/serine-rich Human Tra2 of beta- splicing in recombinant protein, 574- 10 (transformer 2 proteins are globin HeLa confirmed by EMSA HTra2beta1 AAGAA 9546399 578 homolog, sequence- [3043] and S100 and in HeLa nuclear Drosophila), TRA2B, specific constructs nuclear extract and S100. SRFS10, TRAN2B, activators of of murine extracts. EMSA with TRA2-BETA, Htra2- pre-mRNA IgM-based recombinant protein. beta, splicing. pre-mRNA DKFZp686F18120. Cell. 93(1): for in vitro 139-148. splicing.

GGGGAGGGGAGGATGTTAGTATTTACAAAAGATGATTTTAAGAACTTCCAAGAGATGAGTTTAAGAATTCCATAG AGTATTAGTTGTTCACTGTGTAATTAATCCTTCCGGAGAGTCTTTTTTTTTTTTTTTTTAAAGAAACTTTTGGGTGGG TTTTGTTTTTTATTAGTTACCCTAGGGGTATGTTACCCTGGGGTATGAAGGGAGGTGAAGATAACGGAGGGGGGA GAAAAAAAAAAGGAGAAAAAAGGAGCCTAAAATGGGGAATAATTGAAATGGAACAGGGGGTGTGAGGCTGGTT CCTCAGTCCCCATTCCAAACGGAGGATAGAAGCTGTGTATTTATGTGACCTGGCAGATCTCTGGGGCCATAACACT GAAAAGTGAAAGAACCTGGTGGGCAGCTATCTTTGGCTACTGATAACCAGCAGAAATGTCTGTTAATTCTGATTTT CTCAATTTGAAGGGATCAGCTACACTGTTAAATTTTGGAAAGCCACTACCTACTTCCATCAAGTAACTTAGGTTTCG AAATATGGGTTCAACGCACCTCCCTTATTCAAAATGTCAAAATAGATTATTATAATGTATAAAGTAAGAATTGACAA AATATGATTCTTGGGTTGATTGGTCATTTAGAAACTAGCCAAAAGTGAGACTTTTAATGTAGAACATTTTTCAGAAA TGGGTACAAAGAAAAATGCATATTACTGTATATTTCAGAGTGTTTATGTGAACCTTGTATTTAATTGAGAGTCCCAT GTACGTTCTGCAGCCTTTTTGCTGCTTCTATCATCTGAAGTTTGTGT

Bi Splic nd Recognize Article Gene/Construct ing ing Position Protein Name d Protein Notes PubMed ID Reference notes (Target RNA) Assa As Sequence y sa y

SEL EX Fu impo Gene Name nct sing and ion a Synonymous: al select SFRS1, SE ion splicing factor LE of the arginine/serine X const -rich 1 Construct of BRCA1 of Smith PJ, Zhang C, Wang ructs (splicing [672] EX17 - INT17 ran J, Chew SL, Zhang MQ, for factor 2, - EX18 - INT18 - do Krainer AR. (2006) splici alternate EX19 with random m An increased specificity ng splicing 7nt and 14nt inserted 7nt CCUAGG score matrix for the rathe 174-180 SF2/ASF factor), ASF, 16825284 in EX18. Construct an G prediction of SF2/ASF- r than SF2, SF2p33, of SMN1 [6606] d specific exonic splicing for SRp30a, EX6 - INT6 - EX7 - 14 enhancers. bindi MGC5228. INT7 - EX8 with 7nt nt Hum Mol Genet. ng. The shuttling SELEX-winners wit 15(16):2490-2508. Selec protein inserted in EX7. h tion SF2/ASF rec of the binds TAP om const and can bin ructs function as ant in export factors pro HeLa (18364396). tei S100 n. extra ct

175

comp leme nted by reco mbin ant prote in. Winn ers are confi rmed by in vitro splici ng in both HeLa nucle ar extra ct and S100 extra ct comp leme nted by reco mbin ant prote in.

SEL EX impo sing a select ion of the const Fu Gene Name ructs nct and for ion Synonymous: splici al SFRS1, ng SE splicing factor rathe LE arginine/serine r than X -rich 1 Construct of BRCA1 for of Smith PJ, Zhang C, Wang (splicing [672] EX17 - INT17 bindi ran J, Chew SL, Zhang MQ, factor 2, - EX18 - INT18 - ng. do Krainer AR. (2006) alternate EX19 with random Selec m An increased specificity splicing 7nt and 14nt inserted tion 7nt CGGAGG score matrix for the 321-327 SF2/ASF factor), ASF, 16825284 in EX18. Construct of the an A prediction of SF2/ASF- SF2, SF2p33, of SMN1 [6606] const d specific exonic splicing SRp30a, EX6 - INT6 - EX7 - ructs 14 enhancers. MGC5228. INT7 - EX8 with 7nt in nt Hum Mol Genet. The shuttling SELEX-winners HeLa wit 15(16):2490-2508. protein inserted in EX7. S100 h SF2/ASF extra rec binds TAP ct om and can comp bin function as leme ant export factors nted pro (18364396). by tei reco n. mbin ant prote in. Winn ers are confi

176

rmed by in vitro splici ng in both HeLa nucle ar extra ct and S100 extra ct comp leme nted by reco mbin ant prote in.

M uta tio nal an Gene Name aly and sis Synonymous: an SFRS1, d splicing factor pul arginine/serine l- -rich 1 do (splicing wn factor 2, Kammler S, Otte M, In ass alternate Hauber I, Kjems J, Hauber vivo ay splicing J, Schaal H. (2006) splici UGGAAA Constructs of HIV-1 an 489-495 SF2/ASF factor), ASF, 17144911 The strength of the HIV-1 ng in G Tat [155871] EX2. d SF2, SF2p33, 3\' splice sites affects Rev HeLa im SRp30a, function. -T4+ mu MGC5228. Retrovirology. 3:89. cells. no The shuttling blo protein t SF2/ASF wit binds TAP h and can He function as La export factors nu (18364396). cle ar ext rac t.

Gene Name and M Synonymous: uta SFRS1, ge splicing factor nes arginine/serine Haque A, Buratti E, is, -rich 1 Baralle FE. (2010) In we (splicing Functional properties and vivo ste factor 2, evolutionary splicing Constructs of CFTR splici rn alternate constraints on a composite [1080] INT11-EX12- ng blo 686-691 SF2/ASF GGGUAC splicing 19910374 exonic regulatory element INT12. Synthesized assay t, factor), ASF, of splicing in CFTR exon sequences. in siR SF2, SF2p33, 12. HeLa N SRp30a, Nucleic Acids Res. cells. A MGC5228. 38(2):647-659. kn The shuttling oc protein kd SF2/ASF ow binds TAP n and can function as

177

export factors (18364396).

Rooke N, Marko Gene Name vtsov and V, Synonymou Cagavi s: SFRS2, E, splicing Black factor DL. arginine/ser (2003) ine-rich 2, In vitro Roles SC-35, splicing UV crosslink for SR SFRS2A, with and protein SRp30b, Weri-1, immunoprecipit s and Construct of c-src 8-13 SC35 GGAGGA PR264. 12612063 Weri-1 ation with hnRN [20779] EX_N1. SC35 S100 and Weri-1 and P A1 accelerates HeLa HeLa nuclear in the transcriptio nuclear extracts. regulat nal extracts. ion of elongation c-src (co- exon transcriptio N1. nal Mol splicing) Cell (PMID: Biol. 18641664). 23(6): 1874- 1884.

Caputi Gene Name M, and Zahler Synonymou AM. s: SFRS2, (2002) splicing SR factor protein arginine/ser s and ine-rich 2, hnRN SC-35, P H In vitro SFRS2A, regulat Construct of HIV-1 splicing RNA affinity SRp30b, e the env [155971] EX_6D with chromatograph 239-243 SC35 AGGAG PR264. 11847131 splicin and part of flanking HeLa y assay and SC35 g of introns. nuclear immunoblot. accelerates the extracts transcriptio HIV-1 nal tev- elongation specifi (co- c exon transcriptio 6D. nal EMBO splicing) J. (PMID: 21(4): 18641664). 845- 855.

Gene Name Cavalo and c Y, Synonymou Bourg SELEX of 20- s: SFRS2, eois Sequences of 20 nt nt random with splicing CF, random for SELEX. recombinant factor Kister Constructs of protein. arginine/ser L, EXE1A_Adenovirus Winners ine-rich 2, Steven - confirmed by SC-35, in J. partial_INT_E1A_A In vitro EMSA with SFRS2A, (1999) denovirus - splicing recombinant 239-245 SC35 AGGAGAA SRp30b, 10094314 The partial_INT_FN1 - in HeLa protein, UV PR264. splicin EX_ED1_FN1 of S100 crosslink, SC35 g FIBRONECTIN extracts. complementatio accelerates factors (FN1) [2335] for in n assay, transcriptio 9G8 vitro splicing. immunoprecipit nal and Construct of Sp1 unit ations with elongation SRp20 of Adenovirus E1A HeLa S100 and (co- transac for in vitro splicing. nuclear transcriptio tivate extracts. nal splicin splicing) g

178

(PMID: throug 18641664). h differe nt and specifi c enhanc ers. RNA. 5(3): 468- 483.

Caputi Gene Name M, and Zahler Synonymou AM. s: SFRS2, (2002) splicing SR factor protein arginine/ser s and ine-rich 2, hnRN SC-35, P H In vitro SFRS2A, regulat Construct of HIV-1 splicing RNA affinity SRp30b, e the env [155971] EX_6D with chromatograph 249-253 SC35 AGGAG PR264. 11847131 splicin and part of flanking HeLa y assay and SC35 g of introns. nuclear immunoblot. accelerates the extracts transcriptio HIV-1 nal tev- elongation specifi (co- c exon transcriptio 6D. nal EMBO splicing) J. (PMID: 21(4): 18641664). 845- 855.

Rooke N, Marko Gene Name vtsov and V, Synonymou Cagavi s: SFRS2, E, splicing Black factor DL. arginine/ser (2003) ine-rich 2, In vitro Roles SC-35, splicing UV crosslink for SR SFRS2A, with and protein SRp30b, Weri-1, immunoprecipit s and Construct of c-src 322-327 SC35 GGAGGA PR264. 12612063 Weri-1 ation with hnRN [20779] EX_N1. SC35 S100 and Weri-1 and P A1 accelerates HeLa HeLa nuclear in the transcriptio nuclear extracts. regulat nal extracts. ion of elongation c-src (co- exon transcriptio N1. nal Mol splicing) Cell (PMID: Biol. 18641664). 23(6): 1874- 1884.

Gene Name Caputi and M, Synonymou Zahler In vitro s: SFRS2, AM. Construct of HIV-1 splicing RNA affinity splicing (2002) env [155971] EX_6D with chromatograph 329-333 SC35 AGAAG factor 11847131 SR and part of flanking HeLa y assay and arginine/ser protein introns. nuclear immunoblot. ine-rich 2, s and extracts SC-35, hnRN SFRS2A, P H SRp30b, regulat

179

PR264. e the SC35 splicin accelerates g of transcriptio the nal HIV-1 elongation tev- (co- specifi transcriptio c exon nal 6D. splicing) EMBO (PMID: J. 18641664). 21(4): 845- 855.

Caputi Gene Name M, and Zahler Synonymou AM. s: SFRS2, (2002) splicing SR factor protein arginine/ser s and ine-rich 2, hnRN SC-35, P H In vitro SFRS2A, regulat Construct of HIV-1 splicing RNA affinity SRp30b, e the env [155971] EX_6D with chromatograph 426-430 SC35 AGCAG PR264. 11847131 splicin and part of flanking HeLa y assay and SC35 g of introns. nuclear immunoblot. accelerates the extracts transcriptio HIV-1 nal tev- elongation specifi (co- c exon transcriptio 6D. nal EMBO splicing) J. (PMID: 21(4): 18641664). 845- 855.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian Gene Name and Synonymous: LY, Wilson SA, SFRS3, splicing factor Synthesi NMR Allain FH.(2006) arginine/serine-rich 3. zed spectr 87-91 SRp20 UUCAC 17036044 Molecular basis The shuttling protein SRp20 sequenc oscop of RNA binds TAP and can function as es y recognition and export factors (18364396). TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian Gene Name and Synonymous: LY, Wilson SA, SFRS3, splicing factor Synthesi NMR Allain FH.(2006) arginine/serine-rich 3. zed spectr 357-360 SRp20 GAUC 17036044 Molecular basis The shuttling protein SRp20 sequenc oscop of RNA binds TAP and can function as es y recognition and export factors (18364396). TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Gene Name and Synonymous: Hargous Y, Synthesi NMR 467-470 SRp20 GAUC 17036044 SFRS3, splicing factor Hautbergue GM, zed spectr

180

arginine/serine-rich 3. Tintaru AM, sequenc oscop The shuttling protein SRp20 Skrisovska L, es y binds TAP and can function as Golovanov AP, export factors (18364396). Stevenin J, Lian LY, Wilson SA, Allain FH.(2006) Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian Gene Name and Synonymous: LY, Wilson SA, SFRS3, splicing factor Synthesi NMR Allain FH.(2006) arginine/serine-rich 3. zed spectr 510-513 SRp20 CAUC 17036044 Molecular basis The shuttling protein SRp20 sequenc oscop of RNA binds TAP and can function as es y recognition and export factors (18364396). TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian Gene Name and Synonymous: LY, Wilson SA, SFRS3, splicing factor Synthesi NMR Allain FH.(2006) arginine/serine-rich 3. zed spectr 792-796 SRp20 UCAUC 17036044 Molecular basis The shuttling protein SRp20 sequenc oscop of RNA binds TAP and can function as es y recognition and export factors (18364396). TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian Gene Name and Synonymous: LY, Wilson SA, SFRS3, splicing factor Synthesi NMR Allain FH.(2006) arginine/serine-rich 3. zed spectr 793-796 SRp20 CAUC 17036044 Molecular basis The shuttling protein SRp20 sequenc oscop of RNA binds TAP and can function as es y recognition and export factors (18364396). TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Gene Name Liu HX, Construct of SELEX and Zhang M, EX_M1 - INT1 - imposing a UV crosslink, Synonymo Krainer AR. EX_M2 murine selection of the competition and us: SFRS5, (1998) IgM and its constructs for immunoprecipita 7-241 SRp40 AAAGG splicing 9649504 Identification variants splicing rather tion assays in factor of functional obtained by than for binding. HeLa nuclear arginine/ser exonic replacing the Selection of the extracts. ine-rich 5, splicing natural ESE in constructs by HRS. enhancer the M2 exon splicing in HeLa

181

motifs with 20nt S100 extracts recognized by random. complemented individual SR by recombinant proteins. SR protein. Genes Dev. Winners are 12(13): 1998- confirmed by in 2012. vitro splicing in HeLa nuclear extracts.

SELEX Liu HX, imposing a Zhang M, selection of the Krainer AR. constructs for Construct of (1998) splicing rather Gene Name EX_M1 - INT1 - Identification than for binding. and EX_M2 murine of functional Selection of the UV crosslink, Synonymo IgM and its exonic constructs by competition and us: SFRS5, variants splicing splicing in HeLa immunoprecipita 247-251 SRp40 AAAGG splicing 9649504 obtained by enhancer S100 extracts tion assays in factor replacing the motifs complemented HeLa nuclear arginine/ser natural ESE in recognized by by recombinant extracts. ine-rich 5, the M2 exon individual SR SR protein. HRS. with 20nt proteins. Winners are random. Genes Dev. confirmed by in 12(13): 1998- vitro splicing in 2012. HeLa nuclear extracts.

SELEX Liu HX, imposing a Zhang M, selection of the Krainer AR. constructs for Construct of (1998) splicing rather Gene Name EX_M1 - INT1 - Identification than for binding. and EX_M2 murine of functional Selection of the UV crosslink, Synonymo IgM and its exonic constructs by competition and us: SFRS5, variants splicing splicing in HeLa immunoprecipita 281-285 SRp40 ACAGG splicing 9649504 obtained by enhancer S100 extracts tion assays in factor replacing the motifs complemented HeLa nuclear arginine/ser natural ESE in recognized by by recombinant extracts. ine-rich 5, the M2 exon individual SR SR protein. HRS. with 20nt proteins. Winners are random. Genes Dev. confirmed by in 12(13): 1998- vitro splicing in 2012. HeLa nuclear extracts.

SELEX Liu HX, imposing a Zhang M, selection of the Krainer AR. constructs for Construct of (1998) splicing rather Gene Name EX_M1 - INT1 - Identification than for binding. and EX_M2 murine of functional Selection of the UV crosslink, Synonymo IgM and its exonic constructs by competition and us: SFRS5, variants splicing splicing in HeLa immunoprecipita 361-365 SRp40 UCUGG splicing 9649504 obtained by enhancer S100 extracts tion assays in factor replacing the motifs complemented HeLa nuclear arginine/ser natural ESE in recognized by by recombinant extracts. ine-rich 5, the M2 exon individual SR SR protein. HRS. with 20nt proteins. Winners are random. Genes Dev. confirmed by in 12(13): 1998- vitro splicing in 2012. HeLa nuclear extracts.

Liu HX, SELEX Construct of Zhang M, imposing a Gene Name EX_M1 - INT1 - Krainer AR. selection of the and EX_M2 murine (1998) constructs for UV crosslink, Synonymo IgM and its Identification splicing rather competition and us: SFRS5, variants of functional than for binding. immunoprecipita 400-404 SRp40 GCAGC splicing 9649504 obtained by exonic Selection of the tion assays in factor replacing the splicing constructs by HeLa nuclear arginine/ser natural ESE in enhancer splicing in HeLa extracts. ine-rich 5, the M2 exon motifs S100 extracts HRS. with 20nt recognized by complemented random. individual SR by recombinant

182

proteins. SR protein. Genes Dev. Winners are 12(13): 1998- confirmed by in 2012. vitro splicing in HeLa nuclear extracts.

SELEX Liu HX, imposing a Zhang M, selection of the Krainer AR. constructs for Construct of (1998) splicing rather Gene Name EX_M1 - INT1 - Identification than for binding. and EX_M2 murine of functional Selection of the UV crosslink, Synonymo IgM and its exonic constructs by competition and us: SFRS5, variants splicing splicing in HeLa immunoprecipita 771-775 SRp40 GCAGC splicing 9649504 obtained by enhancer S100 extracts tion assays in factor replacing the motifs complemented HeLa nuclear arginine/ser natural ESE in recognized by by recombinant extracts. ine-rich 5, the M2 exon individual SR SR protein. HRS. with 20nt proteins. Winners are random. Genes Dev. confirmed by in 12(13): 1998- vitro splicing in 2012. HeLa nuclear extracts.

SELEX Liu HX, imposing a Zhang M, selection of the Krainer AR. constructs for Construct of (1998) splicing rather Gene Name EX_M1 - INT1 - Identification than for binding. and EX_M2 murine of functional Selection of the UV crosslink, Synonymo IgM and its exonic constructs by competition and us: SFRS5, variants splicing splicing in HeLa immunoprecipita 782-786 SRp40 GCUGC splicing 9649504 obtained by enhancer S100 extracts tion assays in factor replacing the motifs complemented HeLa nuclear arginine/ser natural ESE in recognized by by recombinant extracts. ine-rich 5, the M2 exon individual SR SR protein. HRS. with 20nt proteins. Winners are random. Genes Dev. confirmed by in 12(13): 1998- vitro splicing in 2012. HeLa nuclear extracts.

Liu HX, Zhang M, SELEX imposing a Krainer selection of the AR. (1998) Construct of constructs for Identificati EX_M1 - splicing rather than on of INT1 - for binding. Gene Name and functional EX_M2 Selection of the Synonymous: exonic murine IgM UV crosslink, constructs by SFRS6, splicing splicing and its competition and 107- splicing in HeLa SRp55 UCCGGA factor 9649504 enhancer variants immunoprecipitati 112 S100 extracts arginine/serine- motifs obtained by on assays in HeLa complemented by rich 6, B52, recognized replacing the nuclear extracts. recombinant SR MGC5045. by natural ESE protein. Winners are individual in the M2 confirmed by in SR exon with vitro splicing in proteins. 20nt random. HeLa nuclear Genes Dev. extracts. 12(13): 1998-2012.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and Klarskov K, In vitro SELEX of 20nt random 1 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 0- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGGAU 17548433 1 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 4 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

4 SRp30c AGAAC Gene Name and 17548433 Paradis C, Cloutier Synthesized In vitro SELEX of 20nt random

183

1- Synonymous: P, Shkreta L, oligos. Sequences splicing with recombinant 4 SFRS9, splicing Toutant J, of 20nt random for with HeLa protein. EMSA, UV 5 factor Klarskov K, SELEX. nuclear crosslink, SDS-PAGE arginine/serine- Chabot B. (2007) extracts, with HeLa nuclear rich 9. hnRNP I/PTB can siRNA. extracts. antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, 1 Gene Name and Klarskov K, In vitro SELEX of 20nt random 0 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 9- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c CGGAG 17548433 1 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 1 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 3 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, 2 Gene Name and Klarskov K, In vitro SELEX of 20nt random 1 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 8- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c CGGAG 17548433 2 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 2 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 2 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, 2 Gene Name and Klarskov K, In vitro SELEX of 20nt random 3 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 9- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGGAG 17548433 2 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 4 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 3 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro 2 Gene Name and Antagonistic splicing EMSA using 3 Synonymous: effects of the Construct of assays in recombinant protein. 9- SFRS9, splicing SRp30c protein BCL2L1 [600039] HeLa SRp30c AGGAG 18534987 UV cross-linking in 2 factor and cryptic 5\' EX1-EX2-INT2- nuclear HeLa and 4 arginine/serine- splice sites on the EX3 extracts and immunoprecipitation. 3 rich 9. alternative splicing recombinan of the apoptotic t protein regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, 2 Gene Name and In vitro SELEX of 20nt random Klarskov K, 4 Synonymous: Synthesized splicing with recombinant Chabot B. (2007) 9- SFRS9, splicing oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGGAG 17548433 hnRNP I/PTB can 2 factor of 20nt random for nuclear crosslink, SDS-PAGE antagonize the 5 arginine/serine- SELEX. extracts, with HeLa nuclear splicing repressor 3 rich 9. siRNA. extracts. activity of SRp30c. RNA 13: 1287-

184

1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro 2 Gene Name and Antagonistic splicing EMSA using 4 Synonymous: effects of the Construct of assays in recombinant protein. 9- SFRS9, splicing SRp30c protein BCL2L1 [600039] HeLa SRp30c AGGAG 18534987 UV cross-linking in 2 factor and cryptic 5\' EX1-EX2-INT2- nuclear HeLa and 5 arginine/serine- splice sites on the EX3 extracts and immunoprecipitation. 3 rich 9. alternative splicing recombinan of the apoptotic t protein regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, 3 Gene Name and Klarskov K, In vitro SELEX of 20nt random 2 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 1- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c CGGAG 17548433 3 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 2 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 5 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, 3 Gene Name and Klarskov K, In vitro SELEX of 20nt random 2 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 4- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGGAU 17548433 3 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 2 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 8 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, 3 Gene Name and Klarskov K, In vitro SELEX of 20nt random 8 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 8- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGAAC 17548433 3 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 9 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 2 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, 4 Gene Name and Klarskov K, In vitro SELEX of 20nt random 2 Synonymous: Chabot B. (2007) Synthesized splicing with recombinant 6- SFRS9, splicing hnRNP I/PTB can oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGCAG 17548433 4 factor antagonize the of 20nt random for nuclear crosslink, SDS-PAGE 3 arginine/serine- splicing repressor SELEX. extracts, with HeLa nuclear 0 rich 9. activity of siRNA. extracts. SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier 6 Gene Name and P, Shkreta L, In vitro SELEX of 20nt random 6 Synonymous: Toutant J, Synthesized splicing with recombinant 8- SFRS9, splicing Klarskov K, oligos. Sequences with HeLa protein. EMSA, UV SRp30c AGAAC 17548433 6 factor Chabot B. (2007) of 20nt random for nuclear crosslink, SDS-PAGE 7 arginine/serine- hnRNP I/PTB can SELEX. extracts, with HeLa nuclear 2 rich 9. antagonize the siRNA. extracts. splicing repressor

185

activity of SRp30c. RNA 13: 1287- 1300.

Gene Name and Synonymous: FUSIP1, FUS Ray D, Kazan H, interacting protein Chan ET, Pena (serine/arginine-rich) 1, Castillo L, Chaudhry NSSR, TASR, SRp38, S, Talukder S, TASR1, TASR2, FUSIP2, Blencowe BJ, Morris SFRS13, SRrp40. Q, Hughes TR. Dephosphorylation converts (2009) RNAcompete 135 SRp38 to a splicing Rapid and Synthesized using - SRp38 AAAGAAA repressor (PMID: 19561594 systematic analysis sequences recombinant 141 12419250) of the RNA protein SRp38 is an atypical SR recognition protein that functions as a specificities of general splicing repressor RNA-binding when dephosphorylated, but proteins. when phosphorylated it Nat Biotechnol. functions as a sequence- 27(7):667-670. specific splicing activator (PMID: 18794844).

Gene Name and Synonymous: FUSIP1, FUS Ray D, Kazan H, interacting protein Chan ET, Pena (serine/arginine-rich) 1, Castillo L, Chaudhry NSSR, TASR, SRp38, S, Talukder S, TASR1, TASR2, FUSIP2, Blencowe BJ, Morris SFRS13, SRrp40. Q, Hughes TR. Dephosphorylation converts (2009) RNAcompete 385 SRp38 to a splicing Rapid and Synthesized using - SRp38 GAAAGAA repressor (PMID: 19561594 systematic analysis sequences recombinant 391 12419250) of the RNA protein SRp38 is an atypical SR recognition protein that functions as a specificities of general splicing repressor RNA-binding when dephosphorylated, but proteins. when phosphorylated it Nat Biotechnol. functions as a sequence- 27(7):667-670. specific splicing activator (PMID: 18794844).

Gene Name and Synonymous: FUSIP1, FUS interacting protein (serine/arginine-rich) 1, NSSR, TASR, SRp38, Feng Y, Chen M, TASR1, TASR2, FUSIP2, In vitro Manley JL. (2008) SFRS13, SRrp40. splicing with Phosphorylation EMSA and Dephosphorylation converts HeLa S100 switches the general RNase 603 SRp38 to a splicing Constructs of extracts. splicing repressor protection - SRp38 GACAAA repressor (PMID: 18794844 beta-globin Splicing- SRp38 to a assay with 608 12419250) [3043]. inhibition and sequence-specific recombinant SRp38 is an atypical SR spliceosome- activator. protein. protein that functions as a assembly Nat Struct Mol Biol. general splicing repressor assays. 15(10):1040-1048. when dephosphorylated, but when phosphorylated it functions as a sequence- specific splicing activator (PMID: 18794844).

Gene Name and Ray D, Kazan H, Synonymous: FUSIP1, FUS Chan ET, Pena interacting protein Castillo L, Chaudhry (serine/arginine-rich) 1, S, Talukder S, NSSR, TASR, SRp38, Blencowe BJ, Morris TASR1, TASR2, FUSIP2, Q, Hughes TR. RNAcompete 692 SFRS13, SRrp40. (2009) Synthesized using - SRp38 AAAGAAA 19561594 Dephosphorylation converts Rapid and sequences recombinant 698 SRp38 to a splicing systematic analysis protein repressor (PMID: of the RNA 12419250) recognition SRp38 is an atypical SR specificities of protein that functions as a RNA-binding general splicing repressor proteins.

186

when dephosphorylated, but Nat Biotechnol. when phosphorylated it 27(7):667-670. functions as a sequence- specific splicing activator (PMID: 18794844).

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 40- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 44 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 63- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 67 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 136- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 140 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 387- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 391 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 596- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 600 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

SELEX of 20nt Tacke R, Tohyama M, random with Sequences of 20 nt Ogawa S, Manley JL. recombinant Gene Name and random for SELEX. (1998) protein, Synonymous: Sequence of beta-globin In vitro splicing in 693- Human Tra2 proteins confirmed by HTra2alpha AAGAA TRA2A, 9546399 [3043] and constructs of HeLa S100 and 697 are sequence-specific EMSA in HeLa transformer-2 murine IgM-based pre- nuclear extracts. activators of pre- nuclear extract alpha, HSU53209. mRNA for in vitro mRNA splicing. and S100. EMSA splicing. Cell. 93(1): 139-148. with recombinant protein.

Gene Name and Tacke R, Tohyama Sequences of 20 SELEX of 20nt Synonymous: SFRS10, M, Ogawa S, nt random for In vitro random with splicing factor Manley JL. (1998) SELEX. splicing recombinant 40- arginine/serine-rich 10 Human Tra2 Sequence of in HeLa protein, HTra2beta1 AAGAA 9546399 44 (transformer 2 homolog, proteins are beta-globin S100 and confirmed by Drosophila), TRA2B, sequence-specific [3043] and nuclear EMSA in HeLa SRFS10, TRAN2B, activators of pre- constructs of extracts. nuclear extract TRA2-BETA, Htra2-beta, mRNA splicing. murine IgM- and S100. EMSA

187

DKFZp686F18120. Cell. 93(1): 139- based pre- with recombinant 148. mRNA for in protein. vitro splicing.

Sequences of 20 Tacke R, Tohyama SELEX of 20nt Gene Name and nt random for M, Ogawa S, random with Synonymous: SFRS10, SELEX. Manley JL. (1998) In vitro recombinant splicing factor Sequence of Human Tra2 splicing protein, arginine/serine-rich 10 beta-globin 63- proteins are in HeLa confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 [3043] and 67 sequence-specific S100 and EMSA in HeLa Drosophila), TRA2B, constructs of activators of pre- nuclear nuclear extract SRFS10, TRAN2B, murine IgM- mRNA splicing. extracts. and S100. EMSA TRA2-BETA, Htra2-beta, based pre- Cell. 93(1): 139- with recombinant DKFZp686F18120. mRNA for in 148. protein. vitro splicing.

Sequences of 20 Tacke R, Tohyama SELEX of 20nt Gene Name and nt random for M, Ogawa S, random with Synonymous: SFRS10, SELEX. Manley JL. (1998) In vitro recombinant splicing factor Sequence of Human Tra2 splicing protein, arginine/serine-rich 10 beta-globin 136- proteins are in HeLa confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 [3043] and 140 sequence-specific S100 and EMSA in HeLa Drosophila), TRA2B, constructs of activators of pre- nuclear nuclear extract SRFS10, TRAN2B, murine IgM- mRNA splicing. extracts. and S100. EMSA TRA2-BETA, Htra2-beta, based pre- Cell. 93(1): 139- with recombinant DKFZp686F18120. mRNA for in 148. protein. vitro splicing.

Sequences of 20 Tacke R, Tohyama SELEX of 20nt Gene Name and nt random for M, Ogawa S, random with Synonymous: SFRS10, SELEX. Manley JL. (1998) In vitro recombinant splicing factor Sequence of Human Tra2 splicing protein, arginine/serine-rich 10 beta-globin 387- proteins are in HeLa confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 [3043] and 391 sequence-specific S100 and EMSA in HeLa Drosophila), TRA2B, constructs of activators of pre- nuclear nuclear extract SRFS10, TRAN2B, murine IgM- mRNA splicing. extracts. and S100. EMSA TRA2-BETA, Htra2-beta, based pre- Cell. 93(1): 139- with recombinant DKFZp686F18120. mRNA for in 148. protein. vitro splicing.

Sequences of 20 Tacke R, Tohyama SELEX of 20nt Gene Name and nt random for M, Ogawa S, random with Synonymous: SFRS10, SELEX. Manley JL. (1998) In vitro recombinant splicing factor Sequence of Human Tra2 splicing protein, arginine/serine-rich 10 beta-globin 596- proteins are in HeLa confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 [3043] and 600 sequence-specific S100 and EMSA in HeLa Drosophila), TRA2B, constructs of activators of pre- nuclear nuclear extract SRFS10, TRAN2B, murine IgM- mRNA splicing. extracts. and S100. EMSA TRA2-BETA, Htra2-beta, based pre- Cell. 93(1): 139- with recombinant DKFZp686F18120. mRNA for in 148. protein. vitro splicing.

Gene Name and Tacke R, Tohyama Sequences of 20 In vitro SELEX of 20nt Synonymous: SFRS10, M, Ogawa S, nt random for splicing random with 693- HTra2beta1 AAGAA splicing factor 9546399 Manley JL. (1998) SELEX. in HeLa recombinant 697 arginine/serine-rich 10 Human Tra2 Sequence of S100 and protein, (transformer 2 homolog, proteins are beta-globin nuclear confirmed by

188

Drosophila), TRA2B, sequence-specific [3043] and extracts. EMSA in HeLa SRFS10, TRAN2B, activators of pre- constructs of nuclear extract TRA2-BETA, Htra2-beta, mRNA splicing. murine IgM- and S100. EMSA DKFZp686F18120. Cell. 93(1): 139- based pre- with recombinant 148. mRNA for in protein. vitro splicing.

AGTACAAATAAGGCCTTTGGGATTCTTAATGACATTTATGTTAAAATGTTCTCTTCTCTTTAAACACCGTTTTCCAAT CCACCTGTCAGGGAGTCCAAATCGTGTCTGTGTTGATGATGCTATACTTTGTAGCTAGAAAAACAATTTTAGTGTTG TGGGCTCTGTATTCAGACTTCCTTTTTACAAGACCGATGGGCAGTGATAGATTATTTTATCATATTTAATGCATGGG AAATAGTGTGCTGAGGAAGCTATTAAAAGTATAACTCAGTGAATTGGGTCTGAGTTTTAAATGAGATATTTCAAAA TTGGCTTGCCACTGTAAAAGCGACTAAATAATAATATGATACTGTTCTTTATGATCTTGTCATGTTTCACTGATATGT TTGGGGTCTTCACTATGTAAAAAATGTCAAAATTGTAATGAGCAAGCATGTACAAGTAGTCGTAAATCAAAGGTTT TAAACAGGACTGCATTTTCAATTAGGAAAAGCTGTTTGGCAGATAGCATCCAATGCAAAAACAGAAATATCGTAAC GTTCTGCTTAGTGGGCAAGATAAGATAGGAAAGACATGCTCAAAGAGGCAAAAGAATCATTGCTATCATTCATTCT ACACTAGTTTGAAGAAGTTTTTGTACATCAGAGCACTTCCTTCAGCACACTTTTTTGCCTTCAGATTTCATTTTTTATA AAATGAGAAGACTAATGATAAACTGTAGAAATCAAAATTTATTGAGAAATCTGTTTCTCCTAACAGATAGTAACCC TGCCATGATATACTACTTCAACAATGTTATAAAAGTTATGTGA

Recogniz Splici Article Gene/Construct Binding Position Protein Name ed Protein Notes PubMed ID Reference ng notes (Target RNA) Assay Sequence Assay

Gene Name and Synonymous: SFRS1, splicing factor arginine/serin e-rich 1 Haque A, Buratti E, (splicing Baralle FE. (2010) factor 2, Functional properties In vivo Mutagen Constructs of alternate and evolutionary splicin esis, CFTR [1080] splicing splicing constraints g western INT11-EX12- 20-25 SF2/ASF GGAUUC factor), ASF, 19910374 on a composite assay blot, INT12. SF2, SF2p33, exonic regulatory in siRNA Synthesized SRp30a, element of splicing in HeLa knockdo sequences. MGC5228. CFTR exon 12. cells. wn The shuttling Nucleic Acids Res. protein 38(2):647-659. SF2/ASF binds TAP and can function as export factors (18364396).

Gene Name SELEX and of Synonymous: random SFRS1, 20nt Construct splicing factor with Tacke R, Manley JL. containing In arginine/serin recombi (1995) NCAM1 [4684] vitro e-rich 1 nant The human splicing EX18, splicin (splicing protein. factors ASF/SF2 and downstream 5' g in factor 2, Confirm GGAAA SC35 possess distinct, splice site, alpha HeLa 566-573 SF2/ASF alternate 7543047 ed by GAC functionally globin HBA2 nuclea splicing EMSA, significant RNA [3040] INT2, r and factor), ASF, competit binding specificities. HBA2 EX3. S100 SF2, SF2p33, ion assay EMBO J. 14(14): Sequences of 20 nt extract SRp30a, with 3540-3551. random for s. MGC5228. recombi SELEX. The shuttling nant protein protein SF2/ASF and binds TAP SELEX

189

and can winners. function as UV- export factors crosslink (18364396). ing and immuno precipita tion with Hela nuclear extract or S100 extract.

Gene Name and Synonymous: SFRS2, Constr splicing factor uct of arginine/serin HIV-1 RNA e-rich 2, SC- env Caputi M, Zahler AM. affinity 35, SFRS2A, [15597 (2002) chroma SRp30b, 1] In vitro splicing SR proteins and hnRNP H tograph 441-445 SC35 AGUAG PR264. 11847131 EX_6 with HeLa regulate the splicing of the y assay SC35 D and nuclear extracts HIV-1 tev-specific exon 6D. and accelerates part of EMBO J. 21(4): 845-855. immun transcriptiona flankin oblot. l elongation g (co- introns transcriptiona . l splicing) (PMID: 18641664).

Synthe sized oligos for UV- crossli nk. Gene Name Constr and uct of Synonymous: beta- SFRS2, globin UV splicing factor [3043] crossli arginine/serin Schaal TD, Maniatis T. EX1 - nk and e-rich 2, SC- (1999) INT1 - SDS- 35, SFRS2A, Multiple distinct splicing EX2. PAGE SRp30b, In vitro splicing enhancers in the protein- Constr with 492-498 SC35 AGCUGUU PR264. 9858550 in HeLa S100 coding sequences of a uct HeLa SC35 extracts. constitutively spliced pre- and S100 accelerates mRNA. mutant and transcriptiona Mol Cell Biol. 19(1): 261-73. s of nuclear l elongation beta- extract (co- globin s. transcriptiona [3043] l splicing) EX3 - (PMID: INT3 - 18641664). partial _EX4 - EX2 for in vitro splicin g.

Gene Name Constr and uct of Synonymous: HIV-1 RNA SFRS2, env Caputi M, Zahler AM. affinity splicing factor [15597 (2002) chroma arginine/serin 1] In vitro splicing SR proteins and hnRNP H tograph 627-631 SC35 AGAAG e-rich 2, SC- 11847131 EX_6 with HeLa regulate the splicing of the y assay 35, SFRS2A, D and nuclear extracts HIV-1 tev-specific exon 6D. and SRp30b, part of EMBO J. 21(4): 845-855. immun PR264. flankin oblot. SC35 g accelerates introns transcriptiona .

190

l elongation (co- transcriptiona l splicing) (PMID: 18641664).

Gene Name and Synonymous: SFRS2, Constr splicing factor uct of arginine/serin HIV-1 RNA e-rich 2, SC- env Caputi M, Zahler AM. affinity 35, SFRS2A, [15597 (2002) chroma SRp30b, 1] In vitro splicing SR proteins and hnRNP H tograph 699-703 SC35 AGAAG PR264. 11847131 EX_6 with HeLa regulate the splicing of the y assay SC35 D and nuclear extracts HIV-1 tev-specific exon 6D. and accelerates part of EMBO J. 21(4): 845-855. immun transcriptiona flankin oblot. l elongation g (co- introns transcriptiona . l splicing) (PMID: 18641664).

Hargou s Y, Hautber gue GM, Tintaru AM, Gene Skrisov Name ska L, and Golova Synony nov AP, mous: Steveni SFRS3, n J, splicing Lian factor LY, arginine Wilson /serine- SA, rich 3. Allain The FH.(200 NMR 1-364 SRp20 GAUC shuttlin 17036044 Synthesized sequences 6) spectroscopy g Molecul protein ar basis SRp20 of RNA binds recognit TAP ion and and can TAP function binding as by the export SR factors proteins (183643 SRp20 96). and 9G8. EMBO J. 25(21): 5126- 5137.

Gene Hargou Name s Y, and Hautber Synony gue mous: GM, SFRS3, Tintaru NMR 374-378 SRp20 UUCAC splicing 17036044 AM, Synthesized sequences spectroscopy factor Skrisov arginine ska L, /serine- Golova rich 3. nov AP, The Steveni shuttlin n J,

191

g Lian protein LY, SRp20 Wilson binds SA, TAP Allain and can FH.(200 function 6) as Molecul export ar basis factors of RNA (183643 recognit 96). ion and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21): 5126- 5137.

Hargou s Y, Hautber gue GM, Tintaru AM, Gene Skrisov Name ska L, and Golova Synony nov AP, mous: Steveni SFRS3, n J, splicing Lian factor LY, arginine Wilson /serine- SA, rich 3. Allain The FH.(200 NMR 393-399 SRp20 UCUUCAC shuttlin 17036044 Synthesized sequences 6) spectroscopy g Molecul protein ar basis SRp20 of RNA binds recognit TAP ion and and can TAP function binding as by the export SR factors proteins (183643 SRp20 96). and 9G8. EMBO J. 25(21): 5126- 5137.

Gene Hargou Name s Y, and Hautber Synony gue mous: GM, SFRS3, Tintaru splicing AM, NMR 394-399 SRp20 CUUCAC 17036044 Synthesized sequences factor Skrisov spectroscopy arginine ska L, /serine- Golova rich 3. nov AP, The Steveni shuttlin n J, g Lian

192

protein LY, SRp20 Wilson binds SA, TAP Allain and can FH.(200 function 6) as Molecul export ar basis factors of RNA (183643 recognit 96). ion and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21): 5126- 5137.

Hargou s Y, Hautber gue GM, Tintaru AM, Gene Skrisov Name ska L, and Golova Synony nov AP, mous: Steveni SFRS3, n J, splicing Lian factor LY, arginine Wilson /serine- SA, rich 3. Allain The FH.(200 NMR 395-399 SRp20 UUCAC shuttlin 17036044 Synthesized sequences 6) spectroscopy g Molecul protein ar basis SRp20 of RNA binds recognit TAP ion and and can TAP function binding as by the export SR factors proteins (183643 SRp20 96). and 9G8. EMBO J. 25(21): 5126- 5137.

Gene Hargou Name s Y, and Hautber Synony gue mous: GM, SFRS3, Tintaru splicing AM, NMR 509-512 SRp20 CAUC factor 17036044 Skrisov Synthesized sequences spectroscopy arginine ska L, /serine- Golova rich 3. nov AP, The Steveni shuttlin n J, g Lian protein LY,

193

SRp20 Wilson binds SA, TAP Allain and can FH.(200 function 6) as Molecul export ar basis factors of RNA (183643 recognit 96). ion and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21): 5126- 5137.

Hargou s Y, Hautber gue GM, Tintaru AM, Gene Skrisov Name ska L, and Golova Synony nov AP, mous: Steveni SFRS3, n J, splicing Lian factor LY, arginine Wilson /serine- SA, rich 3. Allain The FH.(200 NMR 640-643 SRp20 CAUC shuttlin 17036044 Synthesized sequences 6) spectroscopy g Molecul protein ar basis SRp20 of RNA binds recognit TAP ion and and can TAP function binding as by the export SR factors proteins (183643 SRp20 96). and 9G8. EMBO J. 25(21): 5126- 5137.

Gene Cavaloc SELEX of 20- Name Y, Sequences of 20 nt nt random and Bourge random for SELEX. with Synony ois CF, Constructs of recombinant mous: Kister EXE1A_Adenovirus - protein. SFRS3, L, partial_INT_E1A_Ade Winners splicing Steveni novirus - In vitro confirmed by UACUUCA factor n J. partial_INT_FN1 - splicing in EMSA with 783-790 SRp20 10094314 A arginine (1999) EX_ED1_FN1 of HeLa S100 recombinant /serine- The FIBRONECTIN (FN1) extracts. protein, UV rich 3. splicing [2335] for in vitro crosslink, The factors splicing. Construct of complementati shuttlin 9G8 Sp1 unit of Adenovirus on assay, g and E1A for in vitro immunoprecip protein SRp20 splicing. itations with SRp20 transact HeLa S100

194

binds ivate and nuclear TAP splicing extracts. and can through function differen as t and export specific factors enhance (183643 rs. 96). RNA. 5(3): 468- 483.

Cavaloc Y, Gene Bourge Name ois CF, and Kister Synony L, SELEX of 20- mous: Steveni nt random SFRS3, n J. Sequences of 20 nt with splicing (1999) random for SELEX. recombinant factor The Constructs of protein. arginine splicing EXE1A_Adenovirus - Winners /serine- factors partial_INT_E1A_Ade confirmed by rich 3. 9G8 novirus - In vitro EMSA with The and partial_INT_FN1 - splicing in recombinant 785-791 SRp20 CUUCAAC shuttlin 10094314 SRp20 EX_ED1_FN1 of HeLa S100 protein, UV g transact FIBRONECTIN (FN1) extracts. crosslink, protein ivate [2335] for in vitro complementati SRp20 splicing splicing. Construct of on assay, binds through Sp1 unit of Adenovirus immunoprecip TAP differen E1A for in vitro itations with and can t and splicing. HeLa S100 function specific and nuclear as enhance extracts. export rs. factors RNA. (183643 5(3): 96). 468- 483.

Hargou s Y, Hautber gue GM, Tintaru Gene AM, Name Skrisov and ska L, Synony Golova mous: nov AP, SFRS3, Steveni splicing n J, factor Lian arginine LY, /serine- Wilson rich 3. SA, The Allain NMR 785-791 SRp20 CUUCAAC shuttlin 17036044 FH.(200 Synthesized sequences spectroscopy g 6) protein Molecul SRp20 ar basis binds of RNA TAP recognit and can ion and function TAP as binding export by the factors SR (183643 proteins 96). SRp20 and 9G8. EMBO J. 25(21): 5126-

195

5137.

SELEX imposing a Liu HX, selection of the Zhang M, constructs for Construct of Krainer AR. splicing rather EX_M1 - (1998) than for INT1 - Identification binding. EX_M2 Gene Name and of functional Selection of the murine IgM UV crosslink, Synonymous: exonic constructs by and its competition and SFRS5, splicing splicing splicing in 4-68 SRp40 ACACC 9649504 variants immunoprecipitation factor enhancer HeLa S100 obtained by assays in HeLa arginine/serine- motifs extracts replacing the nuclear extracts. rich 5, HRS. recognized complemented natural ESE by individual by recombinant in the M2 SR proteins. SR protein. exon with Genes Dev. Winners are 20nt random. 12(13): confirmed by in 1998-2012. vitro splicing in HeLa nuclear extracts.

SELEX imposing a Liu HX, selection of the Zhang M, constructs for Construct of Krainer AR. splicing rather EX_M1 - (1998) than for INT1 - Identification binding. EX_M2 Gene Name and of functional Selection of the murine IgM UV crosslink, Synonymous: exonic constructs by and its competition and SFRS5, splicing splicing splicing in 455-459 SRp40 AAAGG 9649504 variants immunoprecipitation factor enhancer HeLa S100 obtained by assays in HeLa arginine/serine- motifs extracts replacing the nuclear extracts. rich 5, HRS. recognized complemented natural ESE by individual by recombinant in the M2 SR proteins. SR protein. exon with Genes Dev. Winners are 20nt random. 12(13): confirmed by in 1998-2012. vitro splicing in HeLa nuclear extracts.

SELEX imposing a Liu HX, selection of the Zhang M, constructs for Construct of Krainer AR. splicing rather EX_M1 - (1998) than for INT1 - Identification binding. EX_M2 Gene Name and of functional Selection of the murine IgM UV crosslink, Synonymous: exonic constructs by and its competition and SFRS5, splicing splicing splicing in 466-470 SRp40 ACAGG 9649504 variants immunoprecipitation factor enhancer HeLa S100 obtained by assays in HeLa arginine/serine- motifs extracts replacing the nuclear extracts. rich 5, HRS. recognized complemented natural ESE by individual by recombinant in the M2 SR proteins. SR protein. exon with Genes Dev. Winners are 20nt random. 12(13): confirmed by in 1998-2012. vitro splicing in HeLa nuclear extracts.

Liu HX, SELEX Construct of Zhang M, imposing a EX_M1 - Krainer AR. selection of the INT1 - (1998) constructs for EX_M2 Gene Name and Identification splicing rather murine IgM UV crosslink, Synonymous: of functional than for and its competition and SFRS5, splicing exonic binding. 471-475 SRp40 ACUGC 9649504 variants immunoprecipitation factor splicing Selection of the obtained by assays in HeLa arginine/serine- enhancer constructs by replacing the nuclear extracts. rich 5, HRS. motifs splicing in natural ESE recognized HeLa S100 in the M2 by individual extracts exon with SR proteins. complemented 20nt random. Genes Dev. by recombinant

196

12(13): SR protein. 1998-2012. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a Liu HX, selection of the Zhang M, constructs for Construct of Krainer AR. splicing rather EX_M1 - (1998) than for INT1 - Identification binding. EX_M2 Gene Name and of functional Selection of the murine IgM UV crosslink, Synonymous: exonic constructs by and its competition and SFRS5, splicing splicing splicing in 582-586 SRp40 AGAGG 9649504 variants immunoprecipitation factor enhancer HeLa S100 obtained by assays in HeLa arginine/serine- motifs extracts replacing the nuclear extracts. rich 5, HRS. recognized complemented natural ESE by individual by recombinant in the M2 SR proteins. SR protein. exon with Genes Dev. Winners are 20nt random. 12(13): confirmed by in 1998-2012. vitro splicing in HeLa nuclear extracts.

Construct of EX_M1 - SELEX imposing a Liu HX, Zhang M, INT1 - selection of the Krainer AR. EX_M2 constructs for splicing (1998) Gene Name and murine rather than for binding. Identification of UV crosslink, Synonymous: IgM and Selection of the 31 functional exonic competition and SFRS6, splicing its constructs by splicing - splicing enhancer immunoprecipitat SRp55 AUCGUA factor 9649504 variants in HeLa S100 extracts 53 motifs recognized ion assays in arginine/serine- obtained complemented by 6 by individual SR HeLa nuclear rich 6, B52, by recombinant SR proteins. extracts. MGC5045. replacing protein. Winners are Genes Dev. the natural confirmed by in vitro 12(13): 1998- ESE in the splicing in HeLa 2012. M2 exon nuclear extracts. with 20nt random.

Gene Name SELEX and imposing a Synonymous: selection of the SFRS7, constructs for Schaal TD, Maniatis T. splicing factor splicing rather (1999) arginine/serine- Constructs of than for Selection and rich 7 35kDa, Drosophila dsx binding. characterization of pre- SELEX winner AAG3, [40940] EX3 - Selection of the mRNA splicing confermed by 393- HSSG1, INT3 - EX4 constructs by 9G8 UCUUCA 10022858 enhancers: immublotting in 398 RBM37, mutated by splicing in identification of novel Hela nuclear ZCRB2, inserting 18nt HeLa nuclear SR protein-specific extracts. ZCCHC20 randomized in extracts. enhancer sequences. The shuttling EX4. Winners are Mol Cell Biol. 19(3): protein 9G8 confirmed by 1705-1719. binds TAP and in vitro can function as splicing in export factors HeLa S100 (18364396). extracts.

Gene Name and Synonymous: Lynch KW, Maniatis T. SFRS7, (1996) splicing factor Assembly of specific Sequences arginine/serine- SR protein complexes UV-cross link, deriving from 393- rich 7 35kDa, on distinct regulatory immunoprecipitatio 9G8 UCUUCA 8769651 D. 398 AAG3, elements of the n with HeLa melanogaster HSSG1, Drosophila doublesex extracts. dsx [40940]. RBM37, splicing enhancer. ZCRB2, Genes Dev. ZCCHC20 10(16):2089-2101. The shuttling protein 9G8

197

binds TAP and can function as export factors (18364396).

Gene Name and Synonymous: SFRS7, splicing factor Lynch KW, Maniatis T. arginine/serine- (1996) rich 7 35kDa, Assembly of specific Sequences AAG3, SR protein complexes UV-cross link, deriving from 787- HSSG1, on distinct regulatory immunoprecipitatio 9G8 UCAACA 8769651 D. 792 RBM37, elements of the n with HeLa melanogaster ZCRB2, Drosophila doublesex extracts. dsx [40940]. ZCCHC20 splicing enhancer. The shuttling Genes Dev. protein 9G8 10(16):2089-2101. binds TAP and can function as export factors (18364396).

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 185 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AAGAC SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 189 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 246 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGGAA SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 250 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 468 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGGAC SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 472 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, In vitro EMSA using Toutant J, splicing assays recombinant protein. 468 Gene Name and Synonymous: Shkreta L, Construct of BCL2L1 in HeLa 1853498 UV cross-linking in - SRp30c AGGAC SFRS9, splicing factor Goekjian S, [600039] EX1-EX2- nuclear 7 HeLa and 472 arginine/serine-rich 9. Revil T, INT2-EX3 extracts and immunoprecipitation Chabot B. recombinant . (2008) protein

198

Antagonistic effects of the SRp30c protein and cryptic 5\' splice sites on the alternative splicing of the apoptotic regulator Bcl- x. J Biol Chem. 283(31):21315 -21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 486 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGGAA SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 490 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 565 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGGAA SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 569 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 569 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AAGAC SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 573 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 584 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGGCA SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 588 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

199

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 646 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGCAC SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 650 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 658 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AGCAC SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 662 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, SELEX of 20nt Chabot B. In vitro random with 701 Gene Name and Synonymous: (2007) Synthesized oligos. splicing with recombinant protein. 1754843 - SRp30c AAGAC SFRS9, splicing factor hnRNP I/PTB Sequences of 20nt HeLa nuclear EMSA, UV 3 705 arginine/serine-rich 9. can antagonize random for SELEX. extracts, crosslink, SDS- the splicing siRNA. PAGE with HeLa repressor nuclear extracts. activity of SRp30c. RNA 13: 1287- 1300.

Ray D, Kazan H, Gene Name and Chan ET, Synonymous: FUSIP1, Pena FUS interacting protein Castillo L, (serine/arginine-rich) 1, Chaudhry NSSR, TASR, SRp38, S, Talukder TASR1, TASR2, S, FUSIP2, SFRS13, Blencowe SRrp40. BJ, Morris Dephosphorylation Q, Hughes converts SRp38 to a RNAcompete 588 TR. (2009) splicing repressor Synthesized using - SRp38 AAAAGAA 19561594 Rapid and (PMID: 12419250) sequences recombinant 594 systematic SRp38 is an atypical protein analysis of SR protein that the RNA functions as a general recognition splicing repressor when specificities dephosphorylated, but of RNA- when phosphorylated it binding functions as a proteins. sequence-specific Nat splicing activator Biotechnol. (PMID: 18794844). 27(7):667- 670.

Gene Name Wu JY, Kar A, Construct of Positive clones identified by In vivo 626 and Kuo D, Yu B, Tau MAPT fluorescence-activated cell 1694341 splicing - SRp54 AAGAAG Synonymous: Havlioglu N. [4137] EX9 - sorting and visual inspection. 7 in 631 SFRS11, (2006) INT9 - EX10 - Confirmed by UV crosslink, HEK293. splicing factor, SRp54 INT10 - EX11 immunoprecipitation, SDS-

200

arginine/serine- (SFRS11), a with GFP PAGE. rich 11, p54. regulator for cDNA tau exon 10 inserted into alternative EX11. splicing identified by an expression cloning strategy. Mol Cell Biol. 26(18):6739- 6747.

Tacke R, SELEX of Tohyama M, 20nt random Ogawa S, Sequences of 20 with Manley JL. nt random for recombinant (1998) SELEX. Sequence In vitro protein, Gene Name and Human Tra2 of beta-globin splicing in confirmed by AAGA Synonymous: TRA2A, proteins are 590-594 HTra2alpha 9546399 [3043] and HeLa S100 EMSA in A transformer-2 alpha, sequence- constructs of and nuclear HeLa nuclear HSU53209. specific murine IgM-based extracts. extract and activators of pre-mRNA for in S100. EMSA pre-mRNA vitro splicing. with splicing. recombinant Cell. 93(1): protein. 139-148.

Tacke R, SELEX of Tohyama M, 20nt random Ogawa S, Sequences of 20 with Manley JL. nt random for recombinant (1998) SELEX. Sequence In vitro protein, Gene Name and Human Tra2 of beta-globin splicing in confirmed by AAGA Synonymous: TRA2A, proteins are 626-630 HTra2alpha 9546399 [3043] and HeLa S100 EMSA in A transformer-2 alpha, sequence- constructs of and nuclear HeLa nuclear HSU53209. specific murine IgM-based extracts. extract and activators of pre-mRNA for in S100. EMSA pre-mRNA vitro splicing. with splicing. recombinant Cell. 93(1): protein. 139-148.

Tacke R, Sequences Gene Name and Tohyama M, of 20 nt Synonymous: Ogawa S, random for SFRS10, splicing Manley JL. SELEX. SELEX of 20nt factor (1998) Sequence random with arginine/serine-rich Human Tra2 of beta- In vitro splicing recombinant protein, 10 (transformer 2 HTra2beta proteins are globin in HeLa S100 confirmed by EMSA 90-594 AAGAA homolog, 9546399 1 sequence- [3043] and and nuclear in HeLa nuclear Drosophila), specific constructs extracts. extract and S100. TRA2B, SRFS10, activators of of murine EMSA with TRAN2B, TRA2- pre-mRNA IgM-based recombinant protein. BETA, Htra2-beta, splicing. pre-mRNA DKFZp686F18120 Cell. 93(1): for in vitro . 139-148. splicing.

Tsuda K, Someya T, Kuwasako K, Takahashi M, Gene Name and He F, Unzai S, Synonymous: Inoue M, SFRS10, splicing Harada T, factor Watanabe S, arginine/serine-rich Terada T, 10 (transformer 2 Kobayashi N, 625- HTra2beta 2092639 Synthesize GAAGAA homolog, Shirouzu M, NMR spectroscopy 630 1 4 d sequences Drosophila), Kigawa T, TRA2B, SRFS10, Tanaka A, TRAN2B, TRA2- Sugano S, BETA, Htra2-beta, GÃ¼ntert P, DKFZp686F18120 Yokoyama S, . Muto Y.(2010) Structural basis for the dual RNA- recognition

201

modes of human Tra2-Î² RRM. Nucleic Acids Res. 39(4):1538- 1553.

Tacke R, Sequences Gene Name and Tohyama M, of 20 nt Synonymous: Ogawa S, random for SFRS10, splicing Manley JL. SELEX. SELEX of 20nt factor (1998) Sequence random with arginine/serine-rich Human Tra2 of beta- In vitro splicing recombinant protein, 10 (transformer 2 626- HTra2beta proteins are globin in HeLa S100 confirmed by EMSA AAGAA homolog, 9546399 630 1 sequence- [3043] and and nuclear in HeLa nuclear Drosophila), specific constructs extracts. extract and S100. TRA2B, SRFS10, activators of of murine EMSA with TRAN2B, TRA2- pre-mRNA IgM-based recombinant protein. BETA, Htra2-beta, splicing. pre-mRNA DKFZp686F18120 Cell. 93(1): for in vitro . 139-148. splicing.

Wu JY, Kar A, Kuo D, Yu B, Gene Name and Havlioglu N. Synonymous: (2006) Construct SFRS10, splicing SRp54 of Tau Positive clones factor (SFRS11), a MAPT identified by arginine/serine-rich regulator for [4137] EX9 fluorescence- 10 (transformer 2 tau exon 10 - INT9 - activated cell sorting 626- HTra2beta 1694341 In vivo splicing AAGAAG homolog, alternative EX10 - and visual inspection. 631 1 7 in HEK293. Drosophila), splicing INT10 - Confirmed by UV TRA2B, SRFS10, identified by EX11 with crosslink, TRAN2B, TRA2- an expression GFP cDNA immunoprecipitation, BETA, Htra2-beta, cloning inserted SDS-PAGE. DKFZp686F18120 strategy. into EX11. . Mol Cell Biol. 26(18):6739- 6747.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Watanabe S, Gene Name and Terada T, Synonymous: Kobayashi N, SFRS10, splicing Shirouzu M, factor Kigawa T, arginine/serine-rich Tanaka A, 10 (transformer 2 787- HTra2beta 2092639 Sugano S, Synthesize UCAAC homolog, NMR spectroscopy 791 1 4 GÃ¼ntert P, d sequences Drosophila), Yokoyama S, TRA2B, SRFS10, Muto Y.(2010) TRAN2B, TRA2- Structural basis BETA, Htra2-beta, for the dual DKFZp686F18120 RNA- . recognition modes of human Tra2-Î² RRM. Nucleic Acids Res. 39(4):1538- 1553.

TAATATACATTTTAACCTGGGATTTCTAAATTGCTTTAACAAATGCTAATCCTGAGAGTTGCCCTGCAGGACTCAAA AGGGAAAGGTTTTGGGACGTGGCAGAACCCTGCAGGGACATGGAATTAAGGCCATTGCAATGTATCATCTTTGTA GCATTGTCATCACTCCTAAGCTGCCTTCACAGTTTTAGTACACTAAGATGAGGAAATCGAAAATGGGCAGAGAAAG CTCATACTGTATAATTGAAGACAGTGACAGAGAACGTGTCAGTTATGCCAAAACTCTTTTGATTTCTGTTCCAGGAT 202

TTCCAACAAGAGGGGAAAGGAATGACTTGGGAGGGTGGGAAAGACATTAGGAGTTGTTTTTATTTTTTACCTTGG AAGCTTTAGCTACCAATCCAGTACCCTCCTAACTAGAATGTATACACATCAGCAGGACTGACTGACTACTTCATTAG AGATATACTGTACTCATTGGGGGCCTTGGGGGTACTGCTGTTCTTATGTGGGATTTTAATGTTGTAATGTATTGCAT CTTAATGTATTGAATTCATTTTGTTGTACTATATTGGTTGGCATTTTATTAAAATAAATTGTATTGTATCATATTTGTA TGTTTTAAGAGAAAATAATATAAAATACAATATTTGTACTATTATATAGTGCAAAAACTAC

Articl Recognized Protein PubMed Gene/Construct Binding Position Protein Name Reference e Splicing Assay Sequence Notes ID (Target RNA) Assay notes

Gene Name and Synonymous: SFRS1, splicing Liu HX, SELEX imposing a factor Zhang M, selection of the arginine/serin Krainer AR. constructs for e-rich 1 (1998) UV Construct of splicing rather than (splicing Identification crosslink, EX_M1 - INT1 - for binding. factor 2, of functional competition EX_M2 murine Selection of the alternate exonic and IgM and its constructs by splicing splicing immunopre 91-97 SF2/ASF GGGACGU 9649504 variants obtained splicing in HeLa factor), ASF, enhancer cipitation by replacing the S100 extracts SF2, SF2p33, motifs assays in natural ESE in complemented by SRp30a, recognized by HeLa the M2 exon with recombinant SR MGC5228. individual SR nuclear 20nt random. protein. Winners are The shuttling proteins. extracts. confirmed by in vitro protein Genes Dev. splicing in HeLa SF2/ASF 12(13): 1998- nuclear extracts. binds TAP 2012. and can function as export factors (18364396).

Gene Name SELEX of and random Synonymous: 20nt with SFRS1, recombinant splicing Tacke R, protein. factor Manley JL. Confirmed arginine/serin (1995) Construct by EMSA, e-rich 1 The human containing competition (splicing splicing NCAM1 [4684] assay with factor 2, factors EX18, recombinant alternate ASF/SF2 and downstream 5' In vitro splicing in protein and splicing SC35 possess splice site, alpha 324-331 SF2/ASF GGAAUGAC 7543047 HeLa nuclear and SELEX factor), ASF, distinct, globin HBA2 S100 extracts. winners. SF2, SF2p33, functionally [3040] INT2, UV- SRp30a, significant HBA2 EX3. crosslinking MGC5228. RNA binding Sequences of 20 and The shuttling specificities. nt random for immunopre protein EMBO J. SELEX. cipitation SF2/ASF 14(14): 3540- with Hela binds TAP 3551. nuclear and can extract or function as S100 export factors extract. (18364396).

Gene Name Tacke R, SELEX of and Manley JL. Construct random Synonymous: (1995) containing 20nt with SFRS1, The human NCAM1 [4684] recombinant splicing splicing EX18, protein. factor factors downstream 5' Confirmed In vitro splicing in arginine/serin ASF/SF2 and splice site, alpha by EMSA, 343-350 SF2/ASF GGAAAGAC 7543047 HeLa nuclear and e-rich 1 SC35 possess globin HBA2 competition S100 extracts. (splicing distinct, [3040] INT2, assay with factor 2, functionally HBA2 EX3. recombinant alternate significant Sequences of 20 protein and splicing RNA binding nt random for SELEX factor), ASF, specificities. SELEX. winners. SF2, SF2p33, EMBO J. UV-

203

SRp30a, 14(14): 3540- crosslinking MGC5228. 3551. and The shuttling immunopre protein cipitation SF2/ASF with Hela binds TAP nuclear and can extract or function as S100 export factors extract. (18364396).

Gene Name and Synonymous: SFRS1, splicing Liu HX, SELEX imposing a factor Zhang M, selection of the arginine/serin Krainer AR. constructs for e-rich 1 (1998) UV Construct of splicing rather than (splicing Identification crosslink, EX_M1 - INT1 - for binding. factor 2, of functional competition EX_M2 murine Selection of the alternate exonic and IgM and its constructs by splicing splicing immunopre 442-448 SF2/ASF CUGACUA 9649504 variants obtained splicing in HeLa factor), ASF, enhancer cipitation by replacing the S100 extracts SF2, SF2p33, motifs assays in natural ESE in complemented by SRp30a, recognized by HeLa the M2 exon with recombinant SR MGC5228. individual SR nuclear 20nt random. protein. Winners are The shuttling proteins. extracts. confirmed by in vitro protein Genes Dev. splicing in HeLa SF2/ASF 12(13): 1998- nuclear extracts. binds TAP 2012. and can function as export factors (18364396).

Gene Name and Synonymous: Haque A, SFRS1, Buratti E, splicing Baralle FE. factor (2010) arginine/serin Functional e-rich 1 properties and (splicing evolutionary factor 2, splicing Constructs of alternate constraints on CFTR [1080] Mutagenesi splicing a composite INT11-EX12- In vivo splicing s, western 487-492 SF2/ASF GGGUAC 19910374 factor), ASF, exonic INT12. assay in HeLa cells. blot, siRNA SF2, SF2p33, regulatory Synthesized knockdown SRp30a, element of sequences. MGC5228. splicing in The shuttling CFTR exon protein 12. SF2/ASF Nucleic Acids binds TAP Res. and can 38(2):647- function as 659. export factors (18364396).

Gene Name and Synonymous : SFRS2, splicing Caputi M, Zahler factor AM. (2002) Construct of RNA arginine/seri SR proteins and HIV-1 env affinity ne-rich 2, hnRNP H regulate [155971] In vitro splicing chromatog SC-35, 354-358 SC35 AGGAG 11847131 the splicing of the EX_6D and with HeLa raphy SFRS2A, HIV-1 tev- part of nuclear extracts assay and SRp30b, specific exon 6D. flanking immunobl PR264. EMBO J. 21(4): introns. ot. SC35 845-855. accelerates transcription al elongation (co- transcription

204

al splicing) (PMID: 18641664).

Gene Name and Synonymous : SFRS2, splicing factor arginine/seri Caputi M, Zahler ne-rich 2, AM. (2002) Construct of RNA SC-35, SR proteins and HIV-1 env affinity SFRS2A, hnRNP H regulate [155971] In vitro splicing chromatog 431-435 SC35 AGCAG SRp30b, 11847131 the splicing of the EX_6D and with HeLa raphy PR264. HIV-1 tev- part of nuclear extracts assay and SC35 specific exon 6D. flanking immunobl accelerates EMBO J. 21(4): introns. ot. transcription 845-855. al elongation (co- transcription al splicing) (PMID: 18641664).

Gene Name and Synonymous : SFRS2, Synthesized splicing oligos for Schaal TD, factor UV-crosslink. Maniatis T. arginine/seri Construct of (1999) UV ne-rich 2, beta-globin Multiple distinct crosslink SC-35, [3043] EX1 - splicing enhancers and SDS- SFRS2A, INT1 - EX2. In vitro splicing in the protein- PAGE 493-499 SC35 UGCUGUU SRp30b, 9858550 Construct and in HeLa S100 coding sequences with HeLa PR264. mutants of extracts. of a constitutively S100 and SC35 beta-globin spliced pre- nuclear accelerates [3043] EX3 - mRNA. extracts. transcription INT3 - Mol Cell Biol. al elongation partial_EX4 - 19(1): 261-73. (co- EX2 for in transcription vitro splicing. al splicing) (PMID: 18641664).

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 2-146 SRp20 UCAUC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 143-146 SRp20 CAUC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 159-163 SRp20 UCAUC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

160-163 SRp20 CAUC Gene Name and 1703604 Hargous Y, Hautbergue GM, Synthesized NMR

205

Synonymous: SFRS3, 4 Tintaru AM, Skrisovska L, sequences spectroscopy splicing factor Golovanov AP, Stevenin J, Lian arginine/serine-rich 3. LY, Wilson SA, Allain The shuttling protein FH.(2006) SRp20 binds TAP and can Molecular basis of RNA function as export factors recognition and TAP binding by (18364396). the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 177-182 SRp20 CUUCAC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 178-182 SRp20 UUCAC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 427-430 SRp20 CAUC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. 1703604 Synthesized NMR 532-535 SRp20 CAUC FH.(2006) The shuttling protein 4 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binding by function as export factors the SR proteins SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 66-70 SRp40 GCAGG splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Construct Liu HX, Zhang SELEX imposing a of EX_M1 M, Krainer AR. selection of the - INT1 - Gene Name (1998) constructs for splicing EX_M2 and Identification of rather than for binding. murine Synonymo functional Selection of the UV crosslink, IgM and its us: SFRS5, exonic splicing constructs by splicing competition and variants 76-80 SRp40 AAAGG splicing 9649504 enhancer motifs in HeLa S100 extracts immunoprecipitation obtained factor recognized by complemented by assays in HeLa nuclear by arginine/ser individual SR recombinant SR extracts. replacing ine-rich 5, proteins. protein. Winners are the natural HRS. Genes Dev. confirmed by in vitro ESE in the 12(13): 1998- splicing in HeLa M2 exon 2012. nuclear extracts. with 20nt

206

random.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 82-86 SRp40 AAAGG splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 109- SRp40 GCAGG splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation 113 factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 172- SRp40 GCUGC splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation 176 factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 314- SRp40 AGAGG splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation 318 factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Liu HX, Zhang Construct SELEX imposing a M, Krainer AR. of EX_M1 selection of the Gene Name (1998) - INT1 - constructs for splicing and Identification of EX_M2 rather than for binding. Synonymo functional murine Selection of the UV crosslink, us: SFRS5, exonic splicing IgM and its constructs by splicing competition and 321- SRp40 AAAGG splicing 9649504 enhancer motifs variants in HeLa S100 extracts immunoprecipitation 325 factor recognized by obtained complemented by assays in HeLa nuclear arginine/ser individual SR by recombinant SR extracts. ine-rich 5, proteins. replacing protein. Winners are HRS. Genes Dev. the natural confirmed by in vitro 12(13): 1998- ESE in the splicing in HeLa 2012. M2 exon nuclear extracts.

207

with 20nt random.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 432- SRp40 GCAGG splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation 436 factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Construct Liu HX, Zhang of EX_M1 SELEX imposing a M, Krainer AR. - INT1 - selection of the Gene Name (1998) EX_M2 constructs for splicing and Identification of murine rather than for binding. Synonymo functional IgM and its Selection of the UV crosslink, us: SFRS5, exonic splicing variants constructs by splicing competition and 491- SRp40 ACUGC splicing 9649504 enhancer motifs obtained in HeLa S100 extracts immunoprecipitation 495 factor recognized by by complemented by assays in HeLa nuclear arginine/ser individual SR replacing recombinant SR extracts. ine-rich 5, proteins. the natural protein. Winners are HRS. Genes Dev. ESE in the confirmed by in vitro 12(13): 1998- M2 exon splicing in HeLa 2012. with 20nt nuclear extracts. random.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 68- SFRS9, splicing 1754843 with HeLa SRp30c AGGAC hnRNP I/PTB Sequences of EMSA, UV crosslink, 72 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Gene Name and Antagonistic splicing Synonymous: effects of the Construct of assays in EMSA using recombinant 68- SFRS9, splicing 1853498 SRp30c protein BCL2L1 HeLa protein. UV cross-linking SRp30c AGGAC 72 factor 7 and cryptic 5\' [600039] EX1- nuclear in HeLa and arginine/serine- splice sites on EX2-INT2-EX3 extracts and immunoprecipitation. rich 9. the alternative recombinant splicing of the protein apoptotic regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Gene Name and In vitro Shkreta L, Synthesized SELEX of 20nt random Synonymous: splicing Toutant J, oligos. with recombinant protein. 101- SFRS9, splicing 1754843 with HeLa SRp30c AGAAC Klarskov K, Sequences of EMSA, UV crosslink, 105 factor 3 nuclear Chabot B. 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, (2007) SELEX. nuclear extracts. rich 9. siRNA. hnRNP I/PTB can antagonize

208

the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 203- SFRS9, splicing 1754843 with HeLa SRp30c AGGAA hnRNP I/PTB Sequences of EMSA, UV crosslink, 207 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 246- SFRS9, splicing 1754843 with HeLa SRp30c AAGAC hnRNP I/PTB Sequences of EMSA, UV crosslink, 250 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 259- SFRS9, splicing 1754843 with HeLa SRp30c AGAAC hnRNP I/PTB Sequences of EMSA, UV crosslink, 263 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 301- SFRS9, splicing 1754843 with HeLa SRp30c AGGAU hnRNP I/PTB Sequences of EMSA, UV crosslink, 305 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Gene Name and Cloutier P, In vitro Synthesized SELEX of 20nt random Synonymous: Shkreta L, splicing oligos. with recombinant protein. 323- SFRS9, splicing 1754843 Toutant J, with HeLa SRp30c AGGAA Sequences of EMSA, UV crosslink, 327 factor 3 Klarskov K, nuclear 20nt random for SDS-PAGE with HeLa arginine/serine- Chabot B. extracts, SELEX. nuclear extracts. rich 9. (2007) siRNA. hnRNP I/PTB

209

can antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 346- SFRS9, splicing 1754843 with HeLa SRp30c AAGAC hnRNP I/PTB Sequences of EMSA, UV crosslink, 350 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 354- SFRS9, splicing 1754843 with HeLa SRp30c AGGAG hnRNP I/PTB Sequences of EMSA, UV crosslink, 358 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Gene Name and Antagonistic splicing Synonymous: effects of the Construct of assays in EMSA using recombinant 354- SFRS9, splicing 1853498 SRp30c protein BCL2L1 HeLa protein. UV cross-linking SRp30c AGGAG 358 factor 7 and cryptic 5\' [600039] EX1- nuclear in HeLa and arginine/serine- splice sites on EX2-INT2-EX3 extracts and immunoprecipitation. rich 9. the alternative recombinant splicing of the protein apoptotic regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Klarskov K, Gene Name and In vitro Chabot B. Synthesized SELEX of 20nt random Synonymous: splicing (2007) oligos. with recombinant protein. 431- SFRS9, splicing 1754843 with HeLa SRp30c AGCAG hnRNP I/PTB Sequences of EMSA, UV crosslink, 435 factor 3 nuclear can antagonize 20nt random for SDS-PAGE with HeLa arginine/serine- extracts, the splicing SELEX. nuclear extracts. rich 9. siRNA. repressor activity of SRp30c. RNA 13: 1287- 1300.

Gene Name and Paradis C, Synthesized In vitro SELEX of 20nt random 434- Synonymous: 1754843 Cloutier P, oligos. splicing with recombinant protein. SRp30c AGGAC 438 SFRS9, splicing 3 Shkreta L, Sequences of with HeLa EMSA, UV crosslink, factor Toutant J, 20nt random for nuclear SDS-PAGE with HeLa

210

arginine/serine- Klarskov K, SELEX. extracts, nuclear extracts. rich 9. Chabot B. siRNA. (2007) hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Gene Name and Antagonistic splicing Synonymous: effects of the Construct of assays in EMSA using recombinant 434- SFRS9, splicing 1853498 SRp30c protein BCL2L1 HeLa protein. UV cross-linking SRp30c AGGAC 438 factor 7 and cryptic 5\' [600039] EX1- nuclear in HeLa and arginine/serine- splice sites on EX2-INT2-EX3 extracts and immunoprecipitation. rich 9. the alternative recombinant splicing of the protein apoptotic regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Ray D, Kazan H, Chan ET, Pena Castillo L, Gene Name and Synonymous: Chaudhry S, FUSIP1, FUS interacting protein Talukder S, (serine/arginine-rich) 1, NSSR, Blencowe BJ, TASR, SRp38, TASR1, TASR2, Morris Q, FUSIP2, SFRS13, SRrp40. Hughes TR. Dephosphorylation converts SRp38 to RNAcompete 221 (2009) a splicing repressor (PMID: Synthesized using - SRp38 AGAGAAA 19561594 Rapid and 12419250) sequences recombinant 227 systematic SRp38 is an atypical SR protein that protein analysis of the functions as a general splicing RNA repressor when dephosphorylated, but recognition when phosphorylated it functions as a specificities of sequence-specific splicing activator RNA-binding (PMID: 18794844). proteins. Nat Biotechnol. 27(7):667-670.

Ray D, Kazan H, Chan ET, Pena Castillo L, Gene Name and Synonymous: Chaudhry S, FUSIP1, FUS interacting protein Talukder S, (serine/arginine-rich) 1, NSSR, Blencowe BJ, TASR, SRp38, TASR1, TASR2, Morris Q, FUSIP2, SFRS13, SRrp40. Hughes TR. Dephosphorylation converts SRp38 to RNAcompete 621 (2009) a splicing repressor (PMID: Synthesized using - SRp38 AGAGAAA 19561594 Rapid and 12419250) sequences recombinant 627 systematic SRp38 is an atypical SR protein that protein analysis of the functions as a general splicing RNA repressor when dephosphorylated, but recognition when phosphorylated it functions as a specificities of sequence-specific splicing activator RNA-binding (PMID: 18794844). proteins. Nat Biotechnol. 27(7):667-670.

3’ Intron 13 (138758 – 138957)

211

AAATCTGTGCCTCTGCCTCTTGAATTAATTCTTTGGTTGCTTGCATTTGGGAAGGGAATGGAGAAAGGAAAGAACC AATAAAGCTTTCAAAGTTCAAGAAATTCTCCTGTTTGGTCTGCTGCCTTACAACTTAGGTTACTGACAGTTGAGTAA CACAAACACGCCCCCACAAATACATCAATCAAAACTCACACAAAATT

Spli Bindi Recognized Gene/Construct cing Position Protein Name Protein Notes PubMed ID Reference Article notes ng Sequence (Target RNA) Ass Assay ay

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine- NMR Allain FH.(2006) rich 3. Synthesized spectr 176-179 SRp20 CAUC 17036044 Molecular basis The shuttling sequences oscop of RNA protein SRp20 y recognition and binds TAP and TAP binding by can function as the SR proteins export factors SRp20 and 9G8. (18364396). EMBO J. 25(21):5126- 5137.

Constr uct of EX_M SELEX 1 - imposing a INT1 - selection of the EX_M constructs for 2 splicing rather Gene Liu HX, Zhang M, murine than for Name and Krainer AR. (1998) IgM binding. Synonym Identification of and its Selection of the ous: functional exonic variant constructs by UV crosslink, competition SFRS5, 96495 splicing enhancer s splicing in and immunoprecipitation 64-68 SRp40 AAAGG splicing 04 motifs recognized by obtaine HeLa S100 assays in HeLa nuclear factor individual SR d by extracts extracts. arginine/s proteins. replaci complemented erine-rich Genes Dev. 12(13): ng the by recombinant 5, HRS. 1998-2012. natural SR protein. ESE in Winners are the M2 confirmed by in exon vitro splicing in with HeLa nuclear 20nt extracts. random .

Constr uct of EX_M SELEX 1 - imposing a INT1 - selection of the EX_M constructs for 2 splicing rather Gene Liu HX, Zhang M, murine than for Name and Krainer AR. (1998) IgM binding. Synonym Identification of and its Selection of the ous: functional exonic variant constructs by UV crosslink, competition SFRS5, 96495 splicing enhancer s splicing in and immunoprecipitation 118-122 SRp40 GCUGC splicing 04 motifs recognized by obtaine HeLa S100 assays in HeLa nuclear factor individual SR d by extracts extracts. arginine/s proteins. replaci complemented erine-rich Genes Dev. 12(13): ng the by recombinant 5, HRS. 1998-2012. natural SR protein. ESE in Winners are the M2 confirmed by in exon vitro splicing in with HeLa nuclear 20nt extracts. random .

212

Constr uct of EX_M SELEX 1 - imposing a INT1 - selection of the EX_M constructs for 2 splicing rather Gene Liu HX, Zhang M, murine than for Name and Krainer AR. (1998) IgM binding. Synonym Identification of and its Selection of the ous: functional exonic variant constructs by UV crosslink, competition SFRS5, 96495 splicing enhancer s splicing in and immunoprecipitation 159-163 SRp40 ACACG splicing 04 motifs recognized by obtaine HeLa S100 assays in HeLa nuclear factor individual SR d by extracts extracts. arginine/s proteins. replaci complemented erine-rich Genes Dev. 12(13): ng the by recombinant 5, HRS. 1998-2012. natural SR protein. ESE in Winners are the M2 confirmed by in exon vitro splicing in with HeLa nuclear 20nt extracts. random .

Paradis C, Cloutier P, Shkreta L, Toutant J, SELEX of 20nt Gene Name and Klarskov K, random with Synthesized Synonymous: Chabot B. (2007) recombinant oligos. In vitro splicing SFRS9, splicing 1754843 hnRNP I/PTB can protein. EMSA, 66-70 SRp30c AGGAA Sequences of with HeLa nuclear factor 3 antagonize the UV crosslink, 20nt random extracts, siRNA. arginine/serine- splicing repressor SDS-PAGE with for SELEX. rich 9. activity of HeLa nuclear SRp30c. extracts. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, SELEX of 20nt Gene Name and Klarskov K, random with Synthesized Synonymous: Chabot B. (2007) recombinant oligos. In vitro splicing SFRS9, splicing 1754843 hnRNP I/PTB can protein. EMSA, 71-75 SRp30c AGAAC Sequences of with HeLa nuclear factor 3 antagonize the UV crosslink, 20nt random extracts, siRNA. arginine/serine- splicing repressor SDS-PAGE with for SELEX. rich 9. activity of HeLa nuclear SRp30c. extracts. RNA 13: 1287- 1300.

Gene Name and Synonymous: FUSIP1, FUS interacting protein (serine/arginine-rich) 1, NSSR, Ray D, Kazan H, Chan TASR, SRp38, TASR1, TASR2, ET, Pena Castillo L, FUSIP2, SFRS13, SRrp40. Chaudhry S, Talukder S, Dephosphorylation converts SRp38 Blencowe BJ, Morris Q, Synthesi RNAcompete to a splicing repressor (PMID: Hughes TR. (2009) 68- zed using SRp38 GAAAGAA 12419250) 19561594 Rapid and systematic 74 sequenc recombinant SRp38 is an atypical SR protein analysis of the RNA es protein that functions as a general splicing recognition specificities repressor when dephosphorylated, of RNA-binding proteins. but when phosphorylated it Nat Biotechnol. functions as a sequence-specific 27(7):667-670. splicing activator (PMID: 18794844).

Recognize Positio Protein PubMed Article Gene/Construct (Target Splicing Binding Protein Name d Reference n Notes ID notes RNA) Assay Assay Sequence

Tacke R, SELEX of Gene Name Sequences of 20 nt Tohyama M, In vitro 20nt and random for SELEX. Ogawa S, splicing in random Synonymous Sequence of beta-globin Manley JL. HeLa S100 with 70-74 HTra2alpha AAGAA : TRA2A, 9546399 [3043] and constructs of (1998) and recombina transformer- murine IgM-based pre- Human Tra2 nuclear nt protein, 2 alpha, mRNA for in vitro proteins are extracts. confirmed HSU53209. splicing. sequence- by EMSA

213

specific in HeLa activators of nuclear pre-mRNA extract and splicing. S100. Cell. 93(1): EMSA 139-148. with recombina nt protein.

SELEX of Tacke R, 20nt Tohyama M, random Ogawa S, with Manley JL. recombina Gene Name Sequences of 20 nt (1998) In vitro nt protein, and random for SELEX. Human Tra2 splicing in confirmed Synonymous Sequence of beta-globin proteins are HeLa S100 by EMSA 96-100 HTra2alpha AAGAA : TRA2A, 9546399 [3043] and constructs of sequence- and in HeLa transformer- murine IgM-based pre- specific nuclear nuclear 2 alpha, mRNA for in vitro activators of extracts. extract and HSU53209. splicing. pre-mRNA S100. splicing. EMSA Cell. 93(1): with 139-148. recombina nt protein.

Tacke R, Tohyama M, Ogawa S, Manley Sequences of 20 SELEX of 20nt Gene Name and JL. (1998) nt random for random with Synonymous: SFRS10, Human SELEX. recombinant splicing factor Tra2 In vitro Sequence of beta- protein, arginine/serine-rich 10 proteins splicing in 70- globin [3043] and confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 are HeLa S100 74 constructs of EMSA in HeLa Drosophila), TRA2B, sequence- and nuclear murine IgM- nuclear extract SRFS10, TRAN2B, TRA2- specific extracts. based pre-mRNA and S100. EMSA BETA, Htra2-beta, activators for in vitro with recombinant DKFZp686F18120. of pre- splicing. protein. mRNA splicing. Cell. 93(1): 139-148.

Tacke R, Tohyama M, Ogawa S, Manley Sequences of 20 SELEX of 20nt Gene Name and JL. (1998) nt random for random with Synonymous: SFRS10, Human SELEX. recombinant splicing factor Tra2 In vitro Sequence of beta- protein, arginine/serine-rich 10 proteins splicing in 96- globin [3043] and confirmed by HTra2beta1 AAGAA (transformer 2 homolog, 9546399 are HeLa S100 100 constructs of EMSA in HeLa Drosophila), TRA2B, sequence- and nuclear murine IgM- nuclear extract SRFS10, TRAN2B, TRA2- specific extracts. based pre-mRNA and S100. EMSA BETA, Htra2-beta, activators for in vitro with recombinant DKFZp686F18120. of pre- splicing. protein. mRNA splicing. Cell. 93(1): 139-148.

sFlt1_v2 intron 14 (555541–155741)

GTATGGTGGTGGGCACCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGAAGAATCGCTTGAACCCGGAAGGTG GAGATTGCAGTGCGCAGAGATTGCACCATTGCACTCCAGCCTGGGCAACAAGAGCAAAACTCTGTCTCAAAAAAA AAAAAAAAAAAGCTGCCAATCTAAAGATATGATTATTTACTGTGTACCTAG

214

Gene/Constru Bindin Protein Recognized Protein Article Splicin Position PubMed ID Reference ct (Target g Name Sequence Notes notes g Assay RNA) Assay

Gene Name and Synonymous : SFRS1, splicing factor arginine/seri UV ne-rich 1 In vitro crossli (splicing Rooke N, Markovtsov V, splicing nk and factor 2, Cagavi E, Black DL. with immun alternate (2003) Weri-1, opreci splicing Roles for SR proteins and Construct of c- Weri-1 pitatio factor), ASF, 69-74 SF2/ASF AAGGUG 12612063 hnRNP A1 in the src [20779] S100 n with SF2, regulation of c-src exon EX_N1. and Weri-1 SF2p33, N1. HeLa and SRp30a, Mol Cell Biol. 23(6):1874- nuclear HeLa MGC5228. 1884. extracts nuclear The shuttling . extract protein s. SF2/ASF binds TAP and can function as export factors (18364396).

Gen e Na me and Syn ony mou s: SFR S2, spli cing fact or argi nine /seri ne- Caputi M, rich Zahler AM. 2, (2002) RNA SC- SR proteins and affinity Construct of HIV- 35, hnRNP H In vitro chroma 1 env [155971] SFR regulate the splicing with tograph 35-39 SC35 AGGAG 11847131 EX_6D and part S2A splicing of the HeLa nuclear y assay of flanking , HIV-1 tev- extracts and introns. SRp specific exon immun 30b, 6D. oblot. PR2 EMBO J. 21(4): 64. 845-855. SC3 5 acce lerat es tran scri ptio nal elon gati on (co- tran scri ptio nal spli

215

cing ) (PM ID: 186 416 64).

Gen e Na me and Syn ony mou s: SFR S2, spli cing fact or argi nine /seri ne- rich 2, SC- Caputi M, 35, Zahler AM. SFR (2002) RNA S2A SR proteins and affinity , Construct of HIV- hnRNP H In vitro chroma SRp 1 env [155971] regulate the splicing with tograph 48-52 SC35 AGAAG 30b, 11847131 EX_6D and part splicing of the HeLa nuclear y assay PR2 of flanking HIV-1 tev- extracts and 64. introns. specific exon immun SC3 6D. oblot. 5 EMBO J. 21(4): acce 845-855. lerat es tran scri ptio nal elon gati on (co- tran scri ptio nal spli cing ) (PM ID: 186 416 64).

Gen Cavaloc Y, Sequences of 20 nt SELEX e Bourgeois CF, random for of 20- Na Kister L, SELEX. nt me Stevenin J. Constructs of random and (1999) EXE1A_Adenovir with Syn The splicing us - recomb In vitro ony factors 9G8 and partial_INT_E1A_ inant splicing in 73-79 SC35 UGGAGAU mou 10094314 SRp20 Adenovirus - protein. HeLa S100 s: transactivate partial_INT_FN1 - Winner extracts. SFR splicing through EX_ED1_FN1 of s S2, different and FIBRONECTIN confirm spli specific (FN1) [2335] for ed by cing enhancers. in vitro splicing. EMSA fact RNA. 5(3): Construct of Sp1 with or 468-483. unit of Adenovirus recomb

216

argi E1A for in vitro inant nine splicing. protein, /seri UV ne- crosslin rich k, 2, comple SC- mentati 35, on SFR assay, S2A immun , oprecip SRp itations 30b, with PR2 HeLa 64. S100 SC3 and 5 nuclear acce extracts lerat . es tran scri ptio nal elon gati on (co- tran scri ptio nal spli cing ) (PM ID: 186 416 64).

Constru Liu HX, ct of Zhang M, EX_M Krainer 1 - AR. INT1 - (1998) EX_M Identificati 2 SELEX imposing a on of murine selection of the constructs functional UV crosslink, Gene Name IgM for splicing rather than for exonic competition and and its binding. Selection of the splicing and Synonymous: variants constructs by splicing in enhancer immunopreci 61-165 SRp40 GCUGC SFRS5, 9649504 obtaine HeLa S100 extracts motifs pitation splicing factor d by complemented by recognized assays in arginine/serine- replaci recombinant SR protein. by HeLa nuclear rich 5, HRS. ng the Winners are confirmed by individual extracts. natural in vitro splicing in HeLa SR ESE in nuclear extracts. proteins. the M2 Genes exon Dev. with 12(13): 20nt 1998- random 2012. .

Paradis C, Cloutier P, Shkreta L, Toutant Gene Name and J, Klarskov K, SELEX of 20nt random Synthesized In vitro Synonymous: Chabot B. (2007) with recombinant oligos. splicing with SFRS9, splicing hnRNP I/PTB can protein. EMSA, UV 35-39 SRp30c AGGAG 17548433 Sequences of HeLa nuclear factor antagonize the crosslink, SDS-PAGE 20nt random extracts, arginine/serine- splicing repressor with HeLa nuclear for SELEX. siRNA. rich 9. activity of extracts. SRp30c. RNA 13: 1287- 1300.

35-39 SRp30c AGGAG Gene Name and 18534987 Cloutier P, Construct of In vitro EMSA using

217

Synonymous: Toutant J, Shkreta BCL2L1 splicing recombinant protein. SFRS9, splicing L, Goekjian S, [600039] EX1- assays in UV cross-linking in factor Revil T, Chabot B. EX2-INT2- HeLa nuclear HeLa and arginine/serine- (2008) EX3 extracts and immunoprecipitation. rich 9. Antagonistic recombinant effects of the protein SRp30c protein and cryptic 5\' splice sites on the alternative splicing of the apoptotic regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant Gene Name and J, Klarskov K, SELEX of 20nt random Synthesized In vitro Synonymous: Chabot B. (2007) with recombinant oligos. splicing with SFRS9, splicing hnRNP I/PTB can protein. EMSA, UV 44-48 SRp30c AGGCA 17548433 Sequences of HeLa nuclear factor antagonize the crosslink, SDS-PAGE 20nt random extracts, arginine/serine- splicing repressor with HeLa nuclear for SELEX. siRNA. rich 9. activity of extracts. SRp30c. RNA 13: 1287- 1300.

Tacke R, Tohyama M, SELEX of 20nt Sequences of 20 nt Ogawa S, Manley random with Gene Name and random for SELEX. JL. (1998) In vitro recombinant Synonymous: Sequence of beta- Human Tra2 splicing in protein, confirmed TRA2A, globin [3043] and 0-54 HTra2alpha AAGAA 9546399 proteins are HeLa S100 by EMSA in HeLa transformer-2 constructs of murine sequence-specific and nuclear nuclear extract and alpha, IgM-based pre- activators of pre- extracts. S100. EMSA with HSU53209. mRNA for in vitro mRNA splicing. recombinant splicing. Cell. 93(1): 139- protein. 148.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Gene Name and Watanabe S, Terada Synonymous: SFRS10, T, Kobayashi N, splicing factor Shirouzu M, 4 arginine/serine-rich 10 Kigawa T, Tanaka 9- (transformer 2 A, Sugano S, Synthesized NMR HTra2beta1 GAAGAA 20926394 5 homolog, Drosophila), GÃ¼ntert P, sequences spectroscopy 4 TRA2B, SRFS10, Yokoyama S, Muto TRAN2B, TRA2- Y.(2010) BETA, Htra2-beta, Structural basis for DKFZp686F18120. the dual RNA- recognition modes of human Tra2-Î² RRM. Nucleic Acids Res. 39(4):1538-1553.

SELEX of 20nt random Gene Name and Tacke R, Tohyama Sequences of 20 with Synonymous: SFRS10, M, Ogawa S, nt random for In vitro recombinant splicing factor Manley JL. (1998) SELEX. splicing protein, 5 arginine/serine-rich 10 Human Tra2 Sequence of beta- in HeLa confirmed by 0- (transformer 2 proteins are globin [3043] and HTra2beta1 AAGAA 9546399 S100 EMSA in 5 homolog, Drosophila), sequence-specific constructs of and HeLa nuclear 4 TRA2B, SRFS10, activators of pre- murine IgM- nuclear extract and TRAN2B, TRA2- mRNA splicing. based pre-mRNA extracts. S100. EMSA BETA, Htra2-beta, Cell. 93(1): 139- for in vitro with DKFZp686F18120. 148. splicing. recombinant protein.

218

Exon 15a (155742 – 155827)

AACTGTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTCATCATCATCATCATCGTCATCATCATCAT CATCATA

Arti Protein Recognized PubMed cle Gene/Construct Splicing Position Protein Notes Reference Binding Assay Name Sequence ID note (Target RNA) Assay s

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant arginine/serine-rich 3. partial_INT_FN1 - splicing in 10-16 SRp20 CAUCAAC 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 10-13 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 22-25 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 27-31 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 28-31 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, Synonymous: SFRS3, Tintaru AM, Skrisovska L, splicing factor Golovanov AP, Stevenin J, 33-37 SRp20 UCAUC arginine/serine-rich 3. 17036044 Lian LY, Wilson SA, Allain Synthesized sequences NMR spectroscopy The shuttling protein FH.(2006) SRp20 binds TAP and can Molecular basis of RNA function as export factors recognition and TAP

219

(18364396). binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 34-39 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 34-37 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 36-40 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 37-40 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 48-52 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 49-54 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 49-52 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

220

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 50-57 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 51-55 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 52-57 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 52-55 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 53-60 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 54-58 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, Synonymous: SFRS3, Tintaru AM, Skrisovska L, 55-60 SRp20 CAUCAU splicing factor 17036044 Golovanov AP, Stevenin J, Synthesized sequences NMR spectroscopy arginine/serine-rich 3. Lian LY, Wilson SA, Allain The shuttling protein FH.(2006)

221

SRp20 binds TAP and can Molecular basis of RNA function as export factors recognition and TAP (18364396). binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 55-58 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 56-63 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 57-61 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 58-63 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 58-61 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 60-64 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, Synonymous: SFRS3, Tintaru AM, Skrisovska L, 61-64 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy splicing factor Golovanov AP, Stevenin J, arginine/serine-rich 3. Lian LY, Wilson SA, Allain

222

The shuttling protein FH.(2006) SRp20 binds TAP and can Molecular basis of RNA function as export factors recognition and TAP (18364396). binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 66-70 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 67-72 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 67-70 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 68-75 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 69-73 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 70-75 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Gene Name and Hargous Y, Hautbergue GM, 70-73 SRp20 CAUC Synonymous: SFRS3, 17036044 Tintaru AM, Skrisovska L, Synthesized sequences NMR spectroscopy splicing factor Golovanov AP, Stevenin J,

223

arginine/serine-rich 3. Lian LY, Wilson SA, Allain The shuttling protein FH.(2006) SRp20 binds TAP and can Molecular basis of RNA function as export factors recognition and TAP (18364396). binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 71-78 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 72-76 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 73-78 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 73-76 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 74-81 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Gene Name and Hargous Y, Hautbergue GM, Synonymous: SFRS3, Tintaru AM, Skrisovska L, splicing factor Golovanov AP, Stevenin J, arginine/serine-rich 3. Lian LY, Wilson SA, Allain 75-79 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein FH.(2006) SRp20 binds TAP and can Molecular basis of RNA function as export factors recognition and TAP (18364396). binding by the SR proteins

224

SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 76-81 SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 76-79 SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Sequences of 20 nt random for SELEX. Constructs of Gene Name and EXE1A_Adenovirus - Cavaloc Y, Bourgeois CF, Synonymous: SFRS3, partial_INT_E1A_Aden Kister L, Stevenin J. (1999) SELEX of 20-nt random with recombinant protein. splicing factor ovirus - In vitro The splicing factors 9G8 and Winners confirmed by EMSA with recombinant AUCAUCA arginine/serine-rich 3. partial_INT_FN1 - splicing in 77-84 SRp20 10094314 SRp20 transactivate splicing protein, UV crosslink, complementation assay, U The shuttling protein EX_ED1_FN1 of HeLa S100 through different and immunoprecipitations with HeLa S100 and nuclear SRp20 binds TAP and can FIBRONECTIN (FN1) extracts. specific enhancers. extracts. function as export factors [2335] for in vitro RNA. 5(3): 468-483. (18364396). splicing. Construct of Sp1 unit of Adenovirus E1A for in vitro splicing.

Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, splicing factor Lian LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) 78-82 SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy The shuttling protein Molecular basis of RNA SRp20 binds TAP and can recognition and TAP function as export factors binding by the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Hargous Y, Hautbergue GM, Gene Name and Tintaru AM, Skrisovska L, Gene Name and Tintaru AM, Skrisovska L, Synonymous: SFRS3, Golovanov AP, Stevenin J, Synonymous: SFRS3, Golovanov AP, Stevenin J, Lian splicing factor Lian LY, Wilson SA, Allain splicing factor LY, Wilson SA, Allain arginine/serine-rich 3. FH.(2006) NMR spectroscopy 79- arginine/serine-rich 3. Synthesized NMR 79-84 SRp20 CAUCAU 17036044 Synthesized sequences SRp20 CAUC 17036044 FH.(2006) The shuttling protein Molecular basis of RNA 82 The shuttling protein SRp20 sequences spectroscopy Molecular basis of RNA SRp20 binds TAP and can recognition and TAP binds TAP and can function recognition and TAP binding by function as export factors binding by the SR proteins as export factors the SR proteins SRp20 and 9G8. (18364396). SRp20 and 9G8. (18364396). EMBO J. 25(21):5126-5137. EMBO J. 25(21):5126-5137.

Exon 15a 3’UTR (section 1, 155828-156021)

GCTATCATCATTATCATCATCATCATCATCATCATCATAGCTACCATTTATTGAAAACTATTATGTGTCAACTTCAAA GAACTTATCCTTTAGTTGGAGAGCCAAGACAATCATAACAATAACAAATGGCCGGGCATGGTGGCTCACGCCTGT AATCCCAGCACTTTGGGAGGCCAAGGCAGGTGGATCATTTGA

89- Gene Name and Cavaloc Y, Sequences of 20 nt random for In vitro splicing SELEX of 20-nt SRp20 AUCAUCAU 10094314 96 Synonymous: Bourgeois CF, SELEX. Constructs of in HeLa S100 random with

225

SFRS3, splicing Kister L, EXE1A_Adenovirus - extracts. recombinant protein. factor Stevenin J. partial_INT_E1A_Adenovirus - Winners confirmed by arginine/serine-rich (1999) partial_INT_FN1 - EMSA with 3. The splicing EX_ED1_FN1 of recombinant protein, The shuttling protein factors 9G8 and FIBRONECTIN (FN1) [2335] UV crosslink, SRp20 binds TAP SRp20 for in vitro splicing. Construct complementation and can function as transactivate of Sp1 unit of Adenovirus E1A assay, export factors splicing through for in vitro splicing. immunoprecipitations (18364396). different and with HeLa S100 and specific nuclear extracts. enhancers. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 90- SRp20 UCAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 94 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 91- SRp20 CAUCAU 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 96 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 91- SRp20 CAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 94 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Gene Name and Cavaloc Y, SELEX of 20-nt Sequences of 20 nt random for Synonymous: Bourgeois CF, random with SELEX. Constructs of SFRS3, splicing Kister L, recombinant protein. EXE1A_Adenovirus - factor Stevenin J. Winners confirmed by partial_INT_E1A_Adenovirus - arginine/serine-rich (1999) In vitro splicing EMSA with 98- partial_INT_FN1 - SRp20 AUCAUCAU 3. 10094314 The splicing in HeLa S100 recombinant protein, 105 EX_ED1_FN1 of The shuttling protein factors 9G8 and extracts. UV crosslink, FIBRONECTIN (FN1) [2335] SRp20 binds TAP SRp20 complementation for in vitro splicing. Construct and can function as transactivate assay, of Sp1 unit of Adenovirus E1A export factors splicing through immunoprecipitations for in vitro splicing. (18364396). different and with HeLa S100 and

226

specific nuclear extracts. enhancers. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 99- SRp20 UCAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 103 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 100- SRp20 CAUCAU 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 105 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 100- SRp20 CAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 103 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 101- partial_INT_FN1 - recombinant protein, SRp20 AUCAUCAU 3. 10094314 SRp20 in HeLa S100 108 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Gene Name and Hargous Y, 102- Synonymous: Hautbergue GM, SRp20 UCAUC 17036044 Synthesized sequences NMR spectroscopy 106 SFRS3, splicing Tintaru AM, factor Skrisovska L,

227

arginine/serine-rich Golovanov AP, 3. Stevenin J, Lian The shuttling protein LY, Wilson SA, SRp20 binds TAP Allain FH.(2006) and can function as Molecular basis export factors of RNA (18364396). recognition and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 103- SRp20 CAUCAU 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 108 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 103- SRp20 CAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 106 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 104- partial_INT_FN1 - recombinant protein, SRp20 AUCAUCAU 3. 10094314 SRp20 in HeLa S100 111 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Hargous Y, Gene Name and Hautbergue GM, Synonymous: Tintaru AM, SFRS3, splicing Skrisovska L, factor Golovanov AP, arginine/serine-rich Stevenin J, Lian 105- SRp20 UCAUC 3. 17036044 LY, Wilson SA, Synthesized sequences NMR spectroscopy 109 The shuttling protein Allain FH.(2006) SRp20 binds TAP Molecular basis and can function as of RNA export factors recognition and (18364396). TAP binding by the SR proteins

228

SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 106- SRp20 CAUCAU 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 111 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 106- SRp20 CAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 109 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 107- partial_INT_FN1 - recombinant protein, SRp20 AUCAUCAU 3. 10094314 SRp20 in HeLa S100 114 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 108- SRp20 UCAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 112 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Gene Name and Hargous Y, 109- Synonymous: Hautbergue GM, SRp20 CAUCAU 17036044 Synthesized sequences NMR spectroscopy 114 SFRS3, splicing Tintaru AM, factor Skrisovska L,

229

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 109- SRp20 CAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 112 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 110- partial_INT_FN1 - recombinant protein, SRp20 AUCAUCAU 3. 10094314 SRp20 in HeLa S100 117 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 111- SRp20 UCAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 115 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Gene Name and Hautbergue GM, Synonymous: Tintaru AM, SFRS3, splicing Skrisovska L, factor Golovanov AP, arginine/serine-rich Stevenin J, Lian 112- SRp20 CAUCAU 3. 17036044 LY, Wilson SA, Synthesized sequences NMR spectroscopy 117 The shuttling protein Allain FH.(2006) SRp20 binds TAP Molecular basis and can function as of RNA export factors recognition and (18364396). TAP binding by the SR proteins

230

SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 112- SRp20 CAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 115 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 113- partial_INT_FN1 - recombinant protein, SRp20 AUCAUCAU 3. 10094314 SRp20 in HeLa S100 120 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 114- SRp20 UCAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 118 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 115- SRp20 CAUCAU 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 120 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Gene Name and Hargous Y, 115- Synonymous: Hautbergue GM, SRp20 CAUC 17036044 Synthesized sequences NMR spectroscopy 118 SFRS3, splicing Tintaru AM, factor Skrisovska L,

231

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 116- partial_INT_FN1 - recombinant protein, SRp20 AUCAUCAU 3. 10094314 SRp20 in HeLa S100 123 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 117- SRp20 UCAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 121 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 118- SRp20 CAUCAU 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 123 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Hargous Y, Gene Name and Hautbergue GM, Synonymous: Tintaru AM, SFRS3, splicing Skrisovska L, factor Golovanov AP, arginine/serine-rich Stevenin J, Lian 118- SRp20 CAUC 3. 17036044 LY, Wilson SA, Synthesized sequences NMR spectroscopy 121 The shuttling protein Allain FH.(2006) SRp20 binds TAP Molecular basis and can function as of RNA export factors recognition and (18364396). TAP binding by the SR proteins

232

SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

Cavaloc Y, Bourgeois CF, SELEX of 20-nt Gene Name and Kister L, Sequences of 20 nt random for random with Synonymous: Stevenin J. SELEX. Constructs of recombinant protein. SFRS3, splicing (1999) EXE1A_Adenovirus - Winners confirmed by factor The splicing partial_INT_E1A_Adenovirus - EMSA with arginine/serine-rich factors 9G8 and In vitro splicing 151- partial_INT_FN1 - recombinant protein, SRp20 UGUCAAC 3. 10094314 SRp20 in HeLa S100 157 EX_ED1_FN1 of UV crosslink, The shuttling protein transactivate extracts. FIBRONECTIN (FN1) [2335] complementation SRp20 binds TAP splicing through for in vitro splicing. Construct assay, and can function as different and of Sp1 unit of Adenovirus E1A immunoprecipitations export factors specific for in vitro splicing. with HeLa S100 and (18364396). enhancers. nuclear extracts. RNA. 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Gene Name and Skrisovska L, Synonymous: Golovanov AP, SFRS3, splicing Stevenin J, Lian factor LY, Wilson SA, arginine/serine-rich Allain FH.(2006) 271- SRp20 GAUC 3. 17036044 Molecular basis Synthesized sequences NMR spectroscopy 274 The shuttling protein of RNA SRp20 binds TAP recognition and and can function as TAP binding by export factors the SR proteins (18364396). SRp20 and 9G8. EMBO J. 25(21):5126- 5137.

SELEX imposing a selection of the constructs for Liu HX, Zhang splicing rather M, Krainer AR. than for (1998) binding. Identification of Gene Name and Selection of the functional exonic Construct of EX_M1 - INT1 - UV crosslink, Synonymous: constructs by splicing enhancer EX_M2 murine IgM and its competition and 264- SFRS5, splicing splicing in SRp40 GCAGG 9649504 motifs variants obtained by replacing immunoprecipitation 268 factor HeLa S100 recognized by the natural ESE in the M2 exon assays in HeLa nuclear arginine/serine-rich extracts individual SR with 20nt random. extracts. 5, HRS. complemented proteins. by recombinant Genes Dev. SR protein. 12(13): 1998- Winners are 2012. confirmed by in vitro splicing in HeLa nuclear extracts.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and SELEX of 20nt Klarskov K, Synonymous: In vitro splicing random with Chabot B. (2007) 163- SFRS9, splicing Synthesized oligos. Sequences with HeLa recombinant protein. SRp30c AGAAC 17548433 hnRNP I/PTB 167 factor of 20nt random for SELEX. nuclear extracts, EMSA, UV crosslink, can antagonize arginine/serine-rich siRNA. SDS-PAGE with HeLa the splicing 9. nuclear extracts. repressor activity of SRp30c. RNA 13: 1287- 1300.

Gene Name and Paradis C, SELEX of 20nt In vitro splicing Synonymous: Cloutier P, random with 189- Synthesized oligos. Sequences with HeLa SRp30c AAGAC SFRS9, splicing 17548433 Shkreta L, recombinant protein. 193 of 20nt random for SELEX. nuclear extracts, factor Toutant J, EMSA, UV crosslink, siRNA. arginine/serine-rich Klarskov K, SDS-PAGE with HeLa

233

9. Chabot B. (2007) nuclear extracts. hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and SELEX of 20nt Klarskov K, Synonymous: In vitro splicing random with Chabot B. (2007) 245- SFRS9, splicing Synthesized oligos. Sequences with HeLa recombinant protein. SRp30c AGCAC 17548433 hnRNP I/PTB 249 factor of 20nt random for SELEX. nuclear extracts, EMSA, UV crosslink, can antagonize arginine/serine-rich siRNA. SDS-PAGE with HeLa the splicing 9. nuclear extracts. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and SELEX of 20nt Klarskov K, Synonymous: In vitro splicing random with Chabot B. (2007) 262- SFRS9, splicing Synthesized oligos. Sequences with HeLa recombinant protein. SRp30c AGGCA 17548433 hnRNP I/PTB 266 factor of 20nt random for SELEX. nuclear extracts, EMSA, UV crosslink, can antagonize arginine/serine-rich siRNA. SDS-PAGE with HeLa the splicing 9. nuclear extracts. repressor activity of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and SELEX of 20nt Klarskov K, Synonymous: In vitro splicing random with Chabot B. (2007) 269- SFRS9, splicing Synthesized oligos. Sequences with HeLa recombinant protein. SRp30c UGGAU 17548433 hnRNP I/PTB 273 factor of 20nt random for SELEX. nuclear extracts, EMSA, UV crosslink, can antagonize arginine/serine-rich siRNA. SDS-PAGE with HeLa the splicing 9. nuclear extracts. repressor activity of SRp30c. RNA 13: 1287- 1300.

Gene Name and Synonymous: FUSIP1, FUS interacting protein (serine/arginine- rich) 1, NSSR, TASR, SRp38, Feng Y, Chen M, TASR1, TASR2, Manley JL. FUSIP2, SFRS13, (2008) SRrp40. Phosphorylation In vitro splicing Dephosphorylation switches the with HeLa S100 converts SRp38 to a general splicing extracts. EMSA and RNase 190- splicing repressor repressor SRp38 Constructs of beta-globin Splicing- SRp38 AGACAA 18794844 protection assay with 195 (PMID: 12419250) to a sequence- [3043]. inhibition and recombinant protein. SRp38 is an atypical specific spliceosome- SR protein that activator. assembly functions as a Nat Struct Mol assays. general splicing Biol. repressor when 15(10):1040- dephosphorylated, 1048. but when phosphorylated it functions as a sequence-specific splicing activator (PMID: 18794844).

162- HTra2alpha AAGAA Gene Name and 9546399 Tacke R, Sequences of 20 nt random for In vitro splicing SELEX of 20nt

234

166 Synonymous: Tohyama M, SELEX. Sequence of beta- in HeLa S100 random with TRA2A, Ogawa S, globin [3043] and constructs of and nuclear recombinant protein, transformer-2 alpha, Manley JL. murine IgM-based pre-mRNA extracts. confirmed by EMSA HSU53209. (1998) for in vitro splicing. in HeLa nuclear Human Tra2 extract and S100. proteins are EMSA with sequence-specific recombinant protein. activators of pre- mRNA splicing. Cell. 93(1): 139- 148.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Watanabe S, Gene Name and Terada T, Synonymous: Kobayashi N, SFRS10, splicing Shirouzu M, factor Kigawa T, arginine/serine-rich Tanaka A, 12- 10 (transformer 2 HTra2beta1 UCAAC 20926394 Sugano S, Synthesized sequences NMR spectroscopy 16 homolog, GÃ¼ntert P, Drosophila), Yokoyama S, TRA2B, SRFS10, Muto Y.(2010) TRAN2B, TRA2- Structural basis BETA, Htra2-beta, for the dual DKFZp686F18120. RNA-recognition modes of human Tra2-Î² RRM. Nucleic Acids Res. 39(4):1538- 1553.

Tsuda K, Someya T, Kuwasako K, Takahashi M, He F, Unzai S, Inoue M, Harada T, Watanabe S, Gene Name and Terada T, Synonymous: Kobayashi N, SFRS10, splicing Shirouzu M, factor Kigawa T, arginine/serine-rich Tanaka A, 153- 10 (transformer 2 HTra2beta1 UCAAC 20926394 Sugano S, Synthesized sequences NMR spectroscopy 157 homolog, GÃ¼ntert P, Drosophila), Yokoyama S, TRA2B, SRFS10, Muto Y.(2010) TRAN2B, TRA2- Structural basis BETA, Htra2-beta, for the dual DKFZp686F18120. RNA-recognition modes of human Tra2-Î² RRM. Nucleic Acids Res. 39(4):1538- 1553.

Gene Name and Tacke R, Synonymous: Tohyama M, SFRS10, splicing Ogawa S, SELEX of 20nt factor Manley JL. random with Sequences of 20 nt random for arginine/serine-rich (1998) In vitro splicing recombinant protein, SELEX. Sequence of beta- 162- 10 (transformer 2 Human Tra2 in HeLa S100 confirmed by EMSA HTra2beta1 AAGAA 9546399 globin [3043] and constructs of 166 homolog, proteins are and nuclear in HeLa nuclear murine IgM-based pre-mRNA Drosophila), sequence-specific extracts. extract and S100. for in vitro splicing. TRA2B, SRFS10, activators of pre- EMSA with TRAN2B, TRA2- mRNA splicing. recombinant protein. BETA, Htra2-beta, Cell. 93(1): 139- DKFZp686F18120. 148.

Exon15, 3’ UTR (section 2, 156022 – 156308)

235

GGTCAGGAGTTCAAGACCAGCCTGACCAAGATGGTGAAATGCTGTCTCTATTAAAAATACAAAATTAGC CGGGCATGGTGGCTCATGCCTGTAATGCCAGCTACTCGGGAGGCTGAGACAGGAGAATCACTTGAACC CAGGAGGCAGAGGTTGCAGGGAGCCGAGATCGTGTACTGCACTCCAGCCTGGGCAACAAGAGCGAAA CTCCGTCTCAAAAAACAAATAAATAAATAAATAAATAAACAGACAAAATTCACTTTTTATTCTATTAAACT TAACATACATGC

Arti Recognized PubMed cle Gene/Construct Splicing Binding Position Protein Name Protein Notes Reference Sequence ID note (Target RNA) Assay Assay s

SELEX Gene Name and imposing a Synonymous: selection of the SFRS1, constructs for splicing factor splicing rather arginine/serine- than for rich 1 (splicing Liu HX, Zhang M, binding. UV factor 2, Krainer AR. (1998) Selection of crosslink, Construct of EX_M1 alternate Identification of the constructs competiti - INT1 - EX_M2 splicing factor), functional exonic by splicing in on and murine IgM and its ASF, SF2, splicing enhancer HeLa S100 immunop 145-151 SF2/ASF CAGAGGU 9649504 variants obtained by SF2p33, motifs recognized extracts recipitatio replacing the natural SRp30a, by individual SR complemented n assays ESE in the M2 exon MGC5228. proteins. by in HeLa with 20nt random. The shuttling Genes Dev. 12(13): recombinant nuclear protein 1998-2012. SR protein. extracts. SF2/ASF binds Winners are TAP and can confirmed by function as in vitro export factors splicing in (18364396). HeLa nuclear extracts.

Gene Name and Synonymous: SFRS1, splicing factor arginine/serine- Liu HX, Chew SL, rich 1 (splicing Cartegni L, Zhang Constructs of mouse factor 2, MQ, Krainer AR. IgM EX_M1 - INT - alternate (2000) EX_M2 mutated by splicing factor), Exonic splicing inserting 20nt ASF, SF2, enhancer motif 145-151 SF2/ASF CAGAGGU 10629063 randomized in SF2p33, recognized by EX_M2. Construct SRp30a, human SC35 under of mouse IgM MGC5228. splicing EX_C3 - INT - The shuttling conditions. EX_C4. protein Mol Cell Biol. SF2/ASF binds 20(3): 1063-1071. TAP and can function as export factors (18364396).

Gene Name and Synonymous: SFRS1, splicing factor Cartegni L, Krainer arginine/serine- AR. (2002) rich 1 (splicing Disruption of an factor 2, Construct and SF2/ASF- UV alternate mutants of SMN1 dependent exonic crosslink, splicing factor), [6606] and SMN2 In vivo splicing enhancer immunop 244-250 SF2/ASF CAGACAA ASF, SF2, 11925564 [6607] EX6 - splicing in in SMN2 causes recipitatio SF2p33, shortened_INT6 - 293-HEK. spinal muscular n, SDS- SRp30a, EX7 - INT7 - atrophy in the PAGE. MGC5228. shortened_EX8. absence of SMN1. The shuttling Nat Genet. protein 30(4):377-384. SF2/ASF binds TAP and can function as export factors

236

(18364396).

Gene Name and Caputi M, Synonymou Zahler s: SFRS2, AM. splicing (2002) factor SR arginine/seri proteins ne-rich 2, and In vitro SC-35, hnRNP H splicing RNA affinity SFRS2A, regulate Construct of HIV-1 env 1184713 with chromatograph 5-9 SC35 AGGAG SRp30b, the [155971] EX_6D and part of 1 HeLa y assay and PR264. splicing flanking introns. nuclear immunoblot. SC35 of the extracts accelerates HIV-1 transcription tev- al elongation specific (co- exon 6D. transcription EMBO J. al splicing) 21(4): (PMID: 845-855. 18641664).

Gene Name and Caputi M, Synonymou Zahler s: SFRS2, AM. splicing (2002) factor SR arginine/seri proteins ne-rich 2, and In vitro SC-35, hnRNP H splicing RNA affinity SFRS2A, regulate Construct of HIV-1 env 1184713 with chromatograph 120-124 SC35 AGGAG SRp30b, the [155971] EX_6D and part of 1 HeLa y assay and PR264. splicing flanking introns. nuclear immunoblot. SC35 of the extracts accelerates HIV-1 transcription tev- al elongation specific (co- exon 6D. transcription EMBO J. al splicing) 21(4): (PMID: 845-855. 18641664).

Cavaloc Y, Gene Name Bourgeoi and s CF, Synonymou SELEX of 20- Kister L, s: SFRS2, nt random with Stevenin splicing recombinant J. (1999) factor protein. The Sequences of 20 nt random for arginine/seri Winners splicing SELEX. Constructs of ne-rich 2, confirmed by factors EXE1A_Adenovirus - SC-35, In vitro EMSA with 9G8 and partial_INT_E1A_Adenovirus SFRS2A, splicing in recombinant 1009431 SRp20 - partial_INT_FN1 - 120-126 SC35 AGGAGAA SRp30b, HeLa protein, UV 4 transactiv EX_ED1_FN1 of PR264. S100 crosslink, ate FIBRONECTIN (FN1) [2335] SC35 extracts. complementati splicing for in vitro splicing. Construct accelerates on assay, through of Sp1 unit of Adenovirus transcription immunoprecipi different E1A for in vitro splicing. al elongation tations with and (co- HeLa S100 and specific transcription nuclear enhancers al splicing) extracts. . (PMID: RNA. 18641664). 5(3): 468- 483.

Gene Name Caputi M, and Zahler In vitro Synonymou AM. splicing RNA affinity Construct of HIV-1 env s: SFRS2, 1184713 (2002) with chromatograph 139-143 SC35 AGGAG [155971] EX_6D and part of splicing 1 SR HeLa y assay and flanking introns. factor proteins nuclear immunoblot. arginine/seri and extracts ne-rich 2, hnRNP H

237

SC-35, regulate SFRS2A, the SRp30b, splicing PR264. of the SC35 HIV-1 accelerates tev- transcription specific al elongation exon 6D. (co- EMBO J. transcription 21(4): al splicing) 845-855. (PMID: 18641664).

Liu HX, Chew SL, Gene Name Cartegni and L, Zhang Synonymou MQ, s: SFRS2, Krainer splicing AR. factor (2000) arginine/seri Exonic ne-rich 2, splicing Constructs of mouse IgM SC-35, enhancer EX_M1 - INT - EX_M2 SFRS2A, motif 1062906 mutated by inserting 20nt 145-151 SC35 CAGAGGU SRp30b, recognize 3 randomized in EX_M2. PR264. d by Construct of mouse IgM SC35 human EX_C3 - INT - EX_C4. accelerates SC35 transcription under al elongation splicing (co- condition transcription s. al splicing) Mol Cell (PMID: Biol. 18641664). 20(3): 1063- 1071.

Cavaloc Y, Gene Name Bourgeoi and s CF, Synonymou SELEX of 20- Kister L, s: SFRS2, nt random with Stevenin splicing recombinant J. (1999) factor protein. The Sequences of 20 nt random for arginine/seri Winners splicing SELEX. Constructs of ne-rich 2, confirmed by factors EXE1A_Adenovirus - SC-35, In vitro EMSA with 9G8 and partial_INT_E1A_Adenovirus SFRS2A, splicing in recombinant 1009431 SRp20 - partial_INT_FN1 - 156-162 SC35 GGGAGCC SRp30b, HeLa protein, UV 4 transactiv EX_ED1_FN1 of PR264. S100 crosslink, ate FIBRONECTIN (FN1) [2335] SC35 extracts. complementati splicing for in vitro splicing. Construct accelerates on assay, through of Sp1 unit of Adenovirus transcription immunoprecipi different E1A for in vitro splicing. al elongation tations with and (co- HeLa S100 and specific transcription nuclear enhancers al splicing) extracts. . (PMID: RNA. 18641664). 5(3): 468- 483.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Gene Name and Synonymous: Golovanov AP, Stevenin J, SFRS3, splicing factor Lian LY, Wilson SA, Allain Synthesize NMR 165- arginine/serine-rich 3. 1703604 FH.(2006) SRp20 GAUC d spectroscop 168 The shuttling protein SRp20 4 Molecular basis of RNA sequences y binds TAP and can function as recognition and TAP binding export factors (18364396). by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Gene Name and Synonymous: Hargous Y, Hautbergue GM, Synthesize NMR 253- 1703604 SRp20 UUCAC SFRS3, splicing factor Tintaru AM, Skrisovska L, d spectroscop 257 4 arginine/serine-rich 3. Golovanov AP, Stevenin J, sequences y

238

The shuttling protein SRp20 Lian LY, Wilson SA, Allain binds TAP and can function as FH.(2006) export factors (18364396). Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

SELEX imposing a selection of Liu HX, the constructs Zhang M, for splicing Construct of Krainer AR. rather than EX_M1 - (1998) for binding. Gene INT1 - Identification Selection of Name and EX_M2 of functional the constructs Synonymo murine IgM exonic by splicing in UV crosslink, competition us: SFRS5, and its 105- splicing HeLa S100 and immunoprecipitation SRp40 UCGGG splicing 9649504 variants 109 enhancer extracts assays in HeLa nuclear factor obtained by motifs complemente extracts. arginine/se replacing the recognized by d by rine-rich 5, natural ESE in individual SR recombinant HRS. the M2 exon proteins. SR protein. with 20nt Genes Dev. Winners are random. 12(13): 1998- confirmed by 2012. in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of Liu HX, the constructs Zhang M, for splicing Construct of Krainer AR. rather than EX_M1 - (1998) for binding. Gene INT1 - Identification Selection of Name and EX_M2 of functional the constructs Synonymo murine IgM exonic by splicing in UV crosslink, competition us: SFRS5, and its 118- splicing HeLa S100 and immunoprecipitation SRp40 ACAGG splicing 9649504 variants 122 enhancer extracts assays in HeLa nuclear factor obtained by motifs complemente extracts. arginine/se replacing the recognized by d by rine-rich 5, natural ESE in individual SR recombinant HRS. the M2 exon proteins. SR protein. with 20nt Genes Dev. Winners are random. 12(13): 1998- confirmed by 2012. in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of Liu HX, the constructs Zhang M, for splicing Construct of Krainer AR. rather than EX_M1 - (1998) for binding. Gene INT1 - Identification Selection of Name and EX_M2 of functional the constructs Synonymo murine IgM exonic by splicing in UV crosslink, competition us: SFRS5, and its 146- splicing HeLa S100 and immunoprecipitation SRp40 AGAGG splicing 9649504 variants 150 enhancer extracts assays in HeLa nuclear factor obtained by motifs complemente extracts. arginine/se replacing the recognized by d by rine-rich 5, natural ESE in individual SR recombinant HRS. the M2 exon proteins. SR protein. with 20nt Genes Dev. Winners are random. 12(13): 1998- confirmed by 2012. in vitro splicing in HeLa nuclear extracts.

153- Gene Liu HX, Construct of SELEX UV crosslink, competition SRp40 GCAGG 9649504 157 Name and Zhang M, EX_M1 - imposing a and immunoprecipitation

239

Synonymo Krainer AR. INT1 - selection of assays in HeLa nuclear us: SFRS5, (1998) EX_M2 the constructs extracts. splicing Identification murine IgM for splicing factor of functional and its rather than arginine/se exonic variants for binding. rine-rich 5, splicing obtained by Selection of HRS. enhancer replacing the the constructs motifs natural ESE in by splicing in recognized by the M2 exon HeLa S100 individual SR with 20nt extracts proteins. random. complemente Genes Dev. d by 12(13): 1998- recombinant 2012. SR protein. Winners are confirmed by in vitro splicing in HeLa nuclear extracts.

SELEX imposing a selection of Liu HX, the constructs Zhang M, for splicing Construct of Krainer AR. rather than EX_M1 - (1998) for binding. Gene INT1 - Identification Selection of Name and EX_M2 of functional the constructs Synonymo murine IgM exonic by splicing in UV crosslink, competition us: SFRS5, and its 173- splicing HeLa S100 and immunoprecipitation SRp40 ACUGC splicing 9649504 variants 177 enhancer extracts assays in HeLa nuclear factor obtained by motifs complemente extracts. arginine/se replacing the recognized by d by rine-rich 5, natural ESE in individual SR recombinant HRS. the M2 exon proteins. SR protein. with 20nt Genes Dev. Winners are random. 12(13): 1998- confirmed by 2012. in vitro splicing in HeLa nuclear extracts.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro SELEX of 20nt random Klarskov K, Chabot Synthesized Synonymous: splicing with with recombinant B. (2007) oligos. SFRS9, splicing 1754843 HeLa protein. EMSA, UV 5-9 SRp30c AGGAG hnRNP I/PTB can Sequences of factor 3 nuclear crosslink, SDS-PAGE antagonize the 20nt random arginine/serine- extracts, with HeLa nuclear splicing repressor for SELEX. rich 9. siRNA. extracts. activity of SRp30c. RNA 13: 1287-1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Gene Name and Antagonistic effects splicing Construct of EMSA using Synonymous: of the SRp30c protein assays in BCL2L1 recombinant protein. UV SFRS9, splicing 1853498 and cryptic 5\' splice HeLa 5-9 SRp30c AGGAG [600039] EX1- cross-linking in HeLa factor 7 sites on the nuclear EX2-INT2- and arginine/serine- alternative splicing of extracts and EX3 immunoprecipitation. rich 9. the apoptotic recombinant regulator Bcl-x. protein J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro SELEX of 20nt random Klarskov K, Chabot Synthesized Synonymous: splicing with with recombinant B. (2007) oligos. 13- SFRS9, splicing 1754843 HeLa protein. EMSA, UV SRp30c AAGAC hnRNP I/PTB can Sequences of 17 factor 3 nuclear crosslink, SDS-PAGE antagonize the 20nt random arginine/serine- extracts, with HeLa nuclear splicing repressor for SELEX. rich 9. siRNA. extracts. activity of SRp30c. RNA 13: 1287-1300.

240

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro SELEX of 20nt random Klarskov K, Chabot Synthesized Synonymous: splicing with with recombinant B. (2007) oligos. 120- SFRS9, splicing 1754843 HeLa protein. EMSA, UV SRp30c AGGAG hnRNP I/PTB can Sequences of 124 factor 3 nuclear crosslink, SDS-PAGE antagonize the 20nt random arginine/serine- extracts, with HeLa nuclear splicing repressor for SELEX. rich 9. siRNA. extracts. activity of SRp30c. RNA 13: 1287-1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Gene Name and Antagonistic effects splicing Construct of EMSA using Synonymous: of the SRp30c protein assays in BCL2L1 recombinant protein. UV 120- SFRS9, splicing 1853498 and cryptic 5\' splice HeLa SRp30c AGGAG [600039] EX1- cross-linking in HeLa 124 factor 7 sites on the nuclear EX2-INT2- and arginine/serine- alternative splicing of extracts and EX3 immunoprecipitation. rich 9. the apoptotic recombinant regulator Bcl-x. protein J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro SELEX of 20nt random Klarskov K, Chabot Synthesized Synonymous: splicing with with recombinant B. (2007) oligos. 139- SFRS9, splicing 1754843 HeLa protein. EMSA, UV SRp30c AGGAG hnRNP I/PTB can Sequences of 143 factor 3 nuclear crosslink, SDS-PAGE antagonize the 20nt random arginine/serine- extracts, with HeLa nuclear splicing repressor for SELEX. rich 9. siRNA. extracts. activity of SRp30c. RNA 13: 1287-1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. (2008) In vitro Gene Name and Antagonistic effects splicing Construct of EMSA using Synonymous: of the SRp30c protein assays in BCL2L1 recombinant protein. UV 139- SFRS9, splicing 1853498 and cryptic 5\' splice HeLa SRp30c AGGAG [600039] EX1- cross-linking in HeLa 143 factor 7 sites on the nuclear EX2-INT2- and arginine/serine- alternative splicing of extracts and EX3 immunoprecipitation. rich 9. the apoptotic recombinant regulator Bcl-x. protein J Biol Chem. 283(31):21315- 21324.

Paradis C, Cloutier P, Shkreta L, Toutant J, Gene Name and In vitro SELEX of 20nt random Klarskov K, Chabot Synthesized Synonymous: splicing with with recombinant B. (2007) oligos. 142- SFRS9, splicing 1754843 HeLa protein. EMSA, UV SRp30c AGGCA hnRNP I/PTB can Sequences of 146 factor 3 nuclear crosslink, SDS-PAGE antagonize the 20nt random arginine/serine- extracts, with HeLa nuclear splicing repressor for SELEX. rich 9. siRNA. extracts. activity of SRp30c. RNA 13: 1287-1300.

Gene Name and Synonymous: FUSIP1, FUS interacting protein (serine/arginine-rich) 1, NSSR, TASR, SRp38, Feng Y, Chen M, TASR1, TASR2, FUSIP2, Manley JL. (2008) EMSA SFRS13, SRrp40. Phosphorylation and Dephosphorylation switches the general In vitro splicing with RNase Constructs 245 converts SRp38 to a splicing repressor HeLa S100 extracts. protectio of beta- - SRp38 AGACAA splicing repressor (PMID: 18794844 SRp38 to a Splicing-inhibition n assay globin 250 12419250) sequence-specific and spliceosome- with [3043]. SRp38 is an atypical SR activator. assembly assays. recombin protein that functions as a Nat Struct Mol ant general splicing repressor Biol. 15(10):1040- protein. when dephosphorylated, 1048. but when phosphorylated it functions as a sequence- specific splicing activator (PMID: 18794844).

241

Gene Name and Synonymous: FUSIP1, FUS interacting protein (serine/arginine-rich) 1, NSSR, TASR, SRp38, Feng Y, Chen M, TASR1, TASR2, FUSIP2, Manley JL. (2008) EMSA SFRS13, SRrp40. Phosphorylation and Dephosphorylation switches the general In vitro splicing with RNase Constructs 246 converts SRp38 to a splicing repressor HeLa S100 extracts. protectio of beta- - SRp38 GACAAA splicing repressor (PMID: 18794844 SRp38 to a Splicing-inhibition n assay globin 251 12419250) sequence-specific and spliceosome- with [3043]. SRp38 is an atypical SR activator. assembly assays. recombin protein that functions as a Nat Struct Mol ant general splicing repressor Biol. 15(10):1040- protein. when dephosphorylated, 1048. but when phosphorylated it functions as a sequence- specific splicing activator (PMID: 18794844).

Intron 15a (156308 – 156508)

CATTAATTGCCTACTCTGAGCCTGATCCTTTATTATATTCTAGAGAAAATGAAAATAAGCAACACATAATCCTCACCT TTGAAAAACAAACATAATAGGTACAAAGTCAGTTCATAAATGTGCAATGTGAGTGGTATAAAATAACATC

AGATAAAAATTAGCACTTCTGATGACTAATCAGGGGAGATTTCATGGAGGAGA

Recognized Gene/Construct Splicin Bindin Position Protein Name Protein Notes PubMed ID Reference Article notes Sequence (Target RNA) g Assay g Assay

Gene Name and In vitro UV Synonymous: Dye BT, splicing crosslin SFRS1, Buvoli M, and Contructs of k, splicing factor Mayer SA, competi tropomyosin 1 alpha competi arginine/serine Lin CH, tion in TPM1 [7168] EX1 - tion -rich 1 Patton JG. HeLa INT1 - EX2 - INT2 - assay, (splicing factor (1998) cell EX3 with WT and Western 2, alternate Enhancer nuclear mutants competitor blot, splicing elements extracts. sequences of TPM1 EMSA 194-200 SF2/ASF GGAGGAG factor), ASF, 9848651 activate the In vivo EX2 for in vitro using SF2, SF2p33, weak 3\' splicing splicing. Construct of EX2 SRp30a, splice site into TPM1 EX1 - INT1 - alpha- MGC5228. of alpha- smooth EX2 - INT2 - EX3 - TM and The shuttling tropomyosi muscle INT3 - EX4 with wt or purified protein n exon 2. cells mutants EX2 for in proteins SF2/ASF RNA. (SMCs) vivo splicing. or HeLa binds TAP and 4(12): and nuclear can function as 1523-1536. HeLa extracts. export factors cells. (18364396).

Gene Name and Synonymous : SFRS2, Dye BT, Contructs of splicing Buvoli M, tropomyosin 1 alpha In vitro factor Mayer SA, TPM1 [7168] EX1 - splicing and arginine/seri Lin CH, INT1 - EX2 - INT2 - competition ne-rich 2, Patton JG. UV crosslink, EX3 with WT and in HeLa cell SC-35, (1998) competition assay, mutants competitor nuclear SFRS2A, Enhancer Western blot, EMSA GGAGGA sequences of TPM1 extracts. In 194-200 SC35 SRp30b, 9848651 elements using EX2 alpha-TM G EX2 for in vitro vivo PR264. activate the and purified proteins splicing. Construct of splicing into SC35 weak 3\' splice or HeLa nuclear TPM1 EX1 - INT1 - smooth accelerates site of alpha- extracts. EX2 - INT2 - EX3 - muscle cells transcription tropomyosin INT3 - EX4 with wt (SMCs) and al elongation exon 2. or mutants EX2 for in HeLa cells. (co- RNA. 4(12): vivo splicing. transcription 1523-1536. al splicing) (PMID: 18641664).

242

Gene Name and Synonymous : SFRS2, Rooke N, splicing Markovtsov factor V, Cagavi E, arginine/seri Black DL. In vitro ne-rich 2, (2003) splicing SC-35, UV crosslink and Roles for SR with Weri- SFRS2A, immunoprecipitation 1261206 proteins and Construct of c-src 1, Weri-1 194-199 SC35 GGAGGA SRp30b, with Weri-1 and 3 hnRNP A1 in [20779] EX_N1. S100 and PR264. HeLa nuclear the regulation HeLa SC35 extracts. of c-src exon nuclear accelerates N1. extracts. transcription Mol Cell Biol. al elongation 23(6):1874- (co- 1884. transcription al splicing) (PMID: 18641664).

Hallay H, Locker N, Gene Name Ayadi L, and Ropers D, Synonymous Guittet E, : SFRS2, Branlant C. splicing (2006) factor Biochemical arginine/seri and NMR ne-rich 2, study on the SC-35, competition SFRS2A, Sequences deriving Competition assays 1699028 between 195-200 SC35 GAGGAG SRp30b, from HIV-1 Tat with recombinant 1 proteins SC35, PR264. [155871] protein SRp40, and SC35 heterogeneous accelerates nuclear transcription ribonucleoprot al elongation ein A1 at the (co- HIV-1 Tat transcription exon 2 al splicing) splicing site. (PMID: J Biol Chem. 18641664). 281(48):37159 -37174.

Gene Name and Synonymous : SFRS2, splicing Caputi M, factor Zahler AM. arginine/seri (2002) ne-rich 2, SR proteins SC-35, and hnRNP H In vitro Construct of HIV-1 RNA affinity SFRS2A, regulate the splicing 1184713 env [155971] EX_6D chromatography 196-200 SC35 AGGAG SRp30b, splicing of the with HeLa 1 and part of flanking assay and PR264. HIV-1 tev- nuclear introns. immunoblot. SC35 specific exon extracts accelerates 6D. transcription EMBO J. al elongation 21(4): 845- (co- 855. transcription al splicing) (PMID: 18641664).

Hargous Y, Gene Name and Hautbergue GM, Synonymous: SFRS3, Tintaru AM, splicing factor Skrisovska L, Synthesize arginine/serine-rich 3. Golovanov AP, NMR 24-27 SRp20 GAUC 17036044 d The shuttling protein Stevenin J, Lian LY, spectroscopy sequences SRp20 binds TAP and Wilson SA, Allain can function as export FH.(2006) factors (18364396). Molecular basis of RNA recognition and

243

TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Gene Name and Golovanov AP, Synonymous: SFRS3, Stevenin J, Lian LY, splicing factor Wilson SA, Allain Synthesize 145- arginine/serine-rich 3. NMR SRp20 CAUC 17036044 FH.(2006) d 148 The shuttling protein spectroscopy Molecular basis of sequences SRp20 binds TAP and RNA recognition and can function as export TAP binding by the factors (18364396). SR proteins SRp20 and 9G8. EMBO J. 25(21):5126-5137.

Paradis C, Cloutier P, Shkreta L, Gene Name Toutant J, and Synthesize In vitro Klarskov K, Synonymous d oligos. splicing SELEX of 20nt random with Chabot B. (2007) 160- : SFRS9, Sequences with HeLa recombinant protein. EMSA, SRp30c AGCAC 17548433 hnRNP I/PTB 164 splicing of 20nt nuclear UV crosslink, SDS-PAGE can antagonize factor random for extracts, with HeLa nuclear extracts. the splicing arginine/seri SELEX. siRNA. repressor activity ne-rich 9. of SRp30c. RNA 13: 1287- 1300.

Paradis C, Cloutier P, Shkreta L, Gene Name Toutant J, and Synthesize In vitro Klarskov K, Synonymous d oligos. splicing SELEX of 20nt random with Chabot B. (2007) 196- : SFRS9, Sequences with HeLa recombinant protein. EMSA, SRp30c AGGAG 17548433 hnRNP I/PTB 200 splicing of 20nt nuclear UV crosslink, SDS-PAGE can antagonize factor random for extracts, with HeLa nuclear extracts. the splicing arginine/seri SELEX. siRNA. repressor activity ne-rich 9. of SRp30c. RNA 13: 1287- 1300.

Cloutier P, Toutant J, Shkreta L, Goekjian S, Revil T, Chabot B. In vitro Gene Name (2008) splicing and Antagonistic Construct assays in Synonymous effects of the EMSA using recombinant of BCL2L1 HeLa 196- : SFRS9, SRp30c protein protein. UV cross-linking in SRp30c AGGAG 18534987 [600039] nuclear 200 splicing and cryptic 5\' HeLa and EX1-EX2- extracts factor splice sites on the immunoprecipitation. INT2-EX3 and arginine/seri alternative recombinan ne-rich 9. splicing of the t protein apoptotic regulator Bcl-x. J Biol Chem. 283(31):21315- 21324.

Gene Name and Synonymous: FUSIP1, Ray D, Kazan H, FUS interacting protein Chan ET, Pena (serine/arginine-rich) 1, NSSR, TASR, Castillo L, SRp38, TASR1, TASR2, FUSIP2, Chaudhry S, RNAcompete SFRS13, SRrp40. 1956159 Talukder S, Synthesized 42-48 SRp38 AGAGAAA using recombinant Dephosphorylation converts SRp38 to a 4 Blencowe BJ, sequences protein splicing repressor (PMID: 12419250) Morris Q, Hughes SRp38 is an atypical SR protein that TR. (2009) functions as a general splicing repressor Rapid and when dephosphorylated, but when systematic analysis

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phosphorylated it functions as a of the RNA sequence-specific splicing activator recognition (PMID: 18794844). specificities of RNA-binding proteins. Nat Biotechnol. 27(7):667-670.

1. Piva, F., et al., SpliceAid 2: a database of human splicing factors expression data and RNA target motifs. Hum Mutat, 2012. 33(1): p. 81-5.

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APPENDIX E – Materials: Vednros and Catalog Numbers

JEG3 cells (HTB-36), BeWo cells (CCL-98) and Eagle’s Modified Essential Medium (EMEM) (catalog no. 30-2003) and F-12K (catalog no. 30-2004) were obtained from ATCC. Fetal Bovine Serum (FBS) was from Gibco (catalog no. 16141-079). Human umbilical Vein Endothelial Cells, HUVECs (catalog no. CC-2519 & CC-2519A), Endothelial Growth Medium 1, EGM1 (catalog no. CC-4133 and CC-3121), and EGM2 BulletKit (catalog no. CC-3162) were obtained from Lonza. RNeasy Mini Kit was from Qiagen (catalog no. 74106). T75 flasks (catalog no. 430641) and 60mm tissue culture treated plates (catalog no. 430196) were from Corning. Trypsin/EDTA, 0.25%Trypsin/21mM EDTA in HBSS, was from Cellgro (catalog no. 25-053). The High Capacity cDNA RT Kit was from Applied Biosystems (catalog no 438814). Taq PCR Master Mix for endpoint PCR was from Qiagen (catalog no.1007544). Dulbecco’s Phosphate Buffered Saline, DPBS (catalog no. 21-031-CM), was from Cellgro. Agarose powder for gels was from Sigma (A-0169). Qiaex II Gel Extraction Kit was from Qiagen (catalog no. 20021). SYBR Green (catalog no. 4309159) and qPCR reagents targeting 18s (catalog no. 4308329) were from Applied Biosystems. Gentamycin was from CellGro. Taqman Gene Expression Master Mix (catalog no 4369016), Gene Expression Assay Mixes for pan/total Flt1 (catalog no Hs01052961) and full-length Flt1 (catalog no Hs1052944) and custom probes were from Applied Biosystems. AnnexinV/PI staining Binding Buffer (catalog no. V13246) and PacBlue Annexin V conjugate (catalog no A35122) were from Life Technologies. Propidium iodide was from Thermo Fisher (P3566). Inhibtiors SRPIN and TG003 were from Santa Cruz (cat no. sc-394310 and sc-202528, respectively). Beta mercaptoethanol (M-7522), HEPES (H0887), NaF (S-6521) and Na3VO4 (s- 6508) were from Sigma. Glycine (G48-121), HBSS (14175095) Methanol (A452-SK), NaCl (S271-3), Tris (T393-212), Tween20 (BP337) were from Fisher Scientific. EDTA was from Boston Bioproducts, cat no. BM-150

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