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REGULATION OF ALTERNATIVE POLY(A) SITE CHOICE BY CO- TRANSCRIPTIONAL FACTOR RECRUITMENT, POL II PAUSING AND SPLICING By BECKY ANN FUSBY B.S. University of Nebraska Kearney, 2010 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2016 This thesis for the Doctor of Philosophy degree by Becky Ann Fusby has been approved for the Molecular Biology Program by Mair Churchill, Chair Aaron Johnson Jay Hesselberth Richard Davis Dylan Taatjes David Bentley, Advisor Date 05/20/2016 ii Fusby, Becky Ann (Ph.D. Molecular Biology) Regulation of Alternative Poly(A) Site Choice by Co-Transcriptional Factor Recruitment, Pol II Pausing, and Splicing Thesis directed by Professor David L. Bentley ABSTRACT The majority of mammalian genes undergo alternative polyadenylation resulting in production of alternate mRNA isoforms. Aberrant mRNA length due to alternative poly(A) site choice has been associated with numerous diseases, including cancer. For this reason, understanding the mechanistic regulation of poly(A) site choice is critical. The work in this thesis specifically addresses numerous aspects of transcription and mRNA processing that regulate poly(A) site choice. The relationship between a CTD-ligand, its ability to promote Ser2P, and the functional consequence of reduced Ser2P on 3’ UTR poly(A) site choice is examined. Additionally, the importance of poly(A) site processing in promoting pol II pausing, Ser2P, and processing factor recruitment is investigated. Lastly, the regulation of intronic poly(A) sites by splicing factors and functional splicing is analyzed. I find that the extent of Ser2P is not closely coupled to poly(A) site use as predicted, but pol II pausing and processing factor recruitment are directly coupled with poly(A) site utilization. Furthermore, I find that splicing is the major regulator of intronic poly(A) site usage in contradiction to a model of U1 snRNP- specific protection. This research further enhances the understanding of how poly(A) sites are regulated by identifying critical factors that ultimately influence iii alternative polyadenylation. The form and content of this abstract are approved. I recommend its publication. Approved: David Bentley iv To the people who kept me going when I wanted to give up. Thank you for believing in me. v ACKNOWLEDGEMENTS I would like to thank my family and friends for their unconditional love, support, encouragement and sacrifice. I would like to especially thank my parents who gave me the freedom to be myself and pursue my dreams. Their only wish for me has always been my happiness, and for that, I am truly grateful. To my life-partner Jesse, thank you for your love, unwavering support and patience. Thank you for teaching me to worry less and for reminding me to live in the moment. Your love of science and quest for knowledge remind me daily why I pursued this degree. To Stella, thank you for being my support system while finishing my thesis and for being my reality check when I needed it most. I would also like to thank all the people who have helped me along my academic journey. Thank you to my college mentor, Kim Carlson, who taught me not to apologize for who I am and encouraged me to go to graduate school. David, thank you for letting me join your lab and investing your time and energy in training me. I would also like to thank all the present and past members of the Bentley lab for their help and support. To Roberto and Kris, thank you for your friendship both inside and outside the lab. To Tassa, thank you for bringing your cheerfulness and knowledge into the lab. I learned so much from you in a short time. To Michael and Ryan, it has been a pleasure learning and training along side you and I wish you all the luck in your futures. I would also like to thank the BMG department, the Molecular Biology program, and my committee for being passionate and insightful colleagues. vi TABLE OF CONTENTS CHAPTER I. INTRODUCTION…………………………………………………………………….1 1.1 Mechanism of 3’-End Processing……………………………………….... 2 1.1A Properties of Poly(A) Sites………………………………………. 2 CPSF and AAUAAA…………………………………………. 3 CstF and DSE…………………………………………………5 CFI and UGUA……………………………………………….. 6 Additional Cleavage/Polyadenylation Factors……………..7 1.1B APA and Mechanisms of Regulation…………………………… 7 “First Come, First Served” Model…………………………... 8 Survival of the Fittest Model………………………………. 10 Agonist/Antagonist Model…………………………………. 12 1.2 APA Regulation by RNA Polymerase II………………………………… 13 1.2A Regulation by RNA Polymerase II Pausing…………………...14 Promoter Proximal Pause…………………………………. 15 3’-End Pol II Pause………………………………………… 16 1.2B Ser2-CTD Phosphorylation and APA…………………………. 17 Ser2-CTD Kinases and Phosphatases…………………... 18 Phospho-Ser2-CTD Ligands……………………………….21 Integration of Phospho-Ser2-CTD with 3’-End Processing…………………………………………………...22 vii 1.3 Integration of APA and Splicing…………………………………………. 22 1.3A Mechanism of Splicing………………………………………….. 23 Major Spliceosome…………………………………………. 23 1.3B Splicing and Cleavage/Polyadenylation………………………. 25 Interaction of Splicing and Cleavage/Polyadenylation Factors………………………………………………………. 26 IgM Gene: A Balance of Splicing and Polyadenylation …27 U1 snRNP and Polyadenylation.…………………………. 29 1.4 Specific Questions…………………………………………………………30 II. MATERIALS AND METHODS………………………………………………….. 36 2.1 ChIP………………………………………………………………………... 36 2.2 ChIP-seq…………….…………………………………………………….. 37 2.3 Antibodies………………………………………………………………….. 38 2.4 Mini-Gene Transfection…………………………………………………... 38 2.5 RNA Extraction……………………………………………………………. 39 2.6 Poly(A)+ RNA-seq………………………………………………………… 39 2.7 Cell Lines and Growth Conditions………………………………………. 39 2.8 Protein Extractions and Immunoblotting………………………………...40 2.9 3’ RACE……………………………………………………………………. 40 2.10 Real-Time PCR………………………………………………………….. 41 2.11 ASO Transfection………………………………………………………...41 2.12 shRNA-Mediated Spt6 Knockdown……………………………………. 41 viii 2.13 ChIP-seq Pipeline……………………………………………………….. 42 III. THE ROLE OF SPT6 IN REGULATING TRANSCRIPTION, SER2-CTD PHOSPHORYLATION AND MRNA PROCESSING……………44 3.1 Introduction………………………………………………………………… 44 3.2 Results……………………………………………………………………... 48 3.2A Spt6 is Localized at 3’-Ends of Genes in Correlation with Ser2P-CTD………………………………………………………. 48 3.2B Knockdown of Spt6 Through an Inducible shRNA…………... 50 3.2C Spt6 Affects Pol II Distribution at the 5’- and 3’-Ends of Genes…………………………………………………………….. 50 3.2D Spt6 Does Not Generally Affect Pol II Distribution Genome-Wide…………………………………………………… 52 3.2E Spt6 Promotes Ser2-CTD Phosphorylation at 3’-Ends of Genes…………………………………………………………. 52 3.2F Spt6 Does Not Affect Other Phospho-CTD Isoforms……….. 53 3.2G Spt6 Regulates Elongation Complex Factor but Not Ser2 Kinase……………………………………………………… 54 3.2H Spt6 Affects 3’ UTR Alternative Poly(A) Site Choice……….. 56 3.2I Spt6 Alters H3K36me3 on Gene Bodies………………………. 57 3.3 Discussion…………………………………………………………………. 58 IV. COORDINATION OF RNA POLYMERASE II PAUSING AND 3’-END PROCESSING FACTOR RECRUITMENT WITH ALTERNATIVE POLYADENYLATION…………………………………………………………... 74 4.1 Introduction………………………………………………………………… 74 4.2 Results……………………………………………………………………... 77 4.2A Pol II Pause Correlates with Poly(A) Site Usage……………..77 ix 4.2B The μS Poly(A) Site is Necessary for the μS+500 Pause….. 80 4.2C The μS Poly(A) Site is Not Sufficient to Induce Pausing…….81 4.2D CTD Ser2 Phosphorylation is Uncoupled from Pol II Pausing at the μS+500 Site……………………………………. 82 4.2E The β-Globin Poly(A) Site is Necessary for Ser2-CTD Hyperphosphorylation…………………………………………... 83 4.2F Alternative Poly(A) Site Use and CstF Recruitment…………. 84 4.3 Discussion…………………………………………………………………. 85 V. REGULATION OF ALTERNATIVE POLY(A) SITE CHOICE BY SPLICING……..…………………………………………………………………... 98 5.1 Introduction………………………………………………………………… 98 5.2 Results……………………………………………………………………. 103 5.2A snRNA ASOs Attenuate Splicing…………………………….. 103 5.2B Reduction of Splicing Alters Poly(A) Site Use Within Gene Bodies……………………………………………………. 104 5.2C Different Mechanisms of Splicing Inhibition Alternatively Regulate Poly(A) Site Use……………………………………. 106 5.2D U1, U4, and U6 snRNPs Protect Poly(A) Sites Near the TSS…………………………………………………………. 108 5.2E Splicing Preferentially Regulates Use of Intronic Poly(A) Sites……………………………………………………. 109 5.2F snRNP Depletion Favors Intronic Poly(A) Site Use on NR3C1 Mini-Gene……………………………………………... 110 5.2G Attenuation of Splicing by Alternative Mechanisms Increases Intronic Poly(A) Site Use……………………………………… 112 5.2H snRNPs Regulate APA on Integrator Genes in a Potential Negative-Feedback Loop……………………………………... 114 x 5.3 Discussion……………………………………………………………….. 116 VI. CONCLUSIONS………………………………………………………………...128 REFERENCES……………………………………………………………………….. 136 APPENDIX A. CHAPTER III SEQUENCING LIBRARIES…………………………………… 168 B. SPT6 AFFECTED POLY(A) SITES…………………………………………... 169 C.CHAPTER IV SEQUENCING LIBRARIES…………………………………… 172 D. CHAPTER IV PRIMERS………………………………………………………..173 E. CHAPTER V SEQUENCING LIBRARIES…………………………………… 174 F. CHAPTER V PRIMERS………………………………………………………... 175 G. SNRNP AND SSA AFFECTED POLY(A) SITES…………………………… 176 xi LIST OF FIGURES FIGURE 1-1: Diagram of pre-mRNA poly(A) site cis-elements and corresponding factors……………………………………………………………………………… 32 1-2: Density of total pol II, Ser2P- and Ser5P-CTD on a typical mammalian gene…………………………………………………………………. 33 1-3: Schematic of the step-wise major spliceosome reaction……………………. 34 1-4: Diagram of poly(A) site use on the IgM gene and relevant mutants……….
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  • Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1

    Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1

    BIOLOGY OF REPRODUCTION 78, 184–192 (2008) Published online before print 10 October 2007. DOI 10.1095/biolreprod.107.062943 Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1 Sarah E. Fiedler, Malini Bajpai, and Daniel W. Carr2 Department of Medicine, Oregon Health & Sciences University and Veterans Affairs Medical Center, Portland, Oregon 97239 ABSTRACT Guanine nucleotide exchange factors (GEFs) catalyze the GDP for GTP exchange [2]. Activation is negatively regulated by In somatic cells, RHOA mediates actin dynamics through a both guanine nucleotide dissociation inhibitors (RHO GDIs) GNA13-mediated signaling cascade involving RHO kinase and GTPase-activating proteins (GAPs) [1, 2]. Endogenous (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be RHO can be inactivated via C3 exoenzyme ADP-ribosylation, negatively regulated by protein kinase A (PRKA), and it and studies have demonstrated RHO involvement in actin-based interacts with members of the A-kinase anchoring (AKAP) cytoskeletal response to extracellular signals, including lyso- family via intermediary proteins. In spermatozoa, actin poly- merization precedes the acrosome reaction, which is necessary phosphatidic acid (LPA) [2–4]. LPA is known to signal through for normal fertility. The present study was undertaken to G-protein-coupled receptors (GPCRs) [4, 5]; specifically, LPA- determine whether the GNA13-mediated RHOA signaling activated GNA13 (formerly Ga13) promotes RHO activation pathway may be involved in acrosome reaction in bovine through GEFs [4, 6]. Activated RHO-GTP then signals RHO caudal sperm, and whether AKAPs may be involved in its kinase (ROCK), resulting in the phosphorylation and activation targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and of LIM-kinase (LIMK), which in turn phosphorylates and cofilin were all detected by Western blot in bovine caudal inactivates cofilin, an actin depolymerizer, the end result being sperm.
  • B4GALT2 Rabbit Pab

    B4GALT2 Rabbit Pab

    Leader in Biomolecular Solutions for Life Science B4GALT2 Rabbit pAb Catalog No.: A17573 Basic Information Background Catalog No. This gene is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes. They A17573 encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta1,4 linkage to Observed MW similar acceptor sugars: GlcNAc, Glc, and Xyl. Each beta4GalT has a distinct function in 42kDa the biosynthesis of different glycoconjugates and saccharide structures. As type II membrane proteins, they have an N-terminal hydrophobic signal sequence that directs Calculated MW the protein to the Golgi apparatus and which then remains uncleaved to function as a transmembrane anchor. By sequence similarity, the beta4GalTs form four groups: Category beta4GalT1 and beta4GalT2, beta4GalT3 and beta4GalT4, beta4GalT5 and beta4GalT6, and beta4GalT7. The enzyme encoded by this gene synthesizes N-acetyllactosamine in Primary antibody glycolipids and glycoproteins. Its substrate specificity is affected by alpha-lactalbumin but it is not expressed in lactating mammary tissue. Three transcript variants encoding Applications two different isoforms have been found for this gene. [provided by RefSeq, Jul 2011] WB,IHC Cross-Reactivity Human, Mouse, Rat Recommended Dilutions Immunogen Information WB 1:500 - 1:2000 Gene ID Swiss Prot 8704 O60909 IHC 1:100 - 1:200 Immunogen Recombinant fusion protein containing a sequence corresponding to amino acids 155-271 of human B4GALT2 (NP_085076.2). Synonyms B4Gal-T2;B4Gal-T3;beta4Gal-T2;B4GALT2 Contact Product Information 400-999-6126 Source Isotype Purification Rabbit IgG Affinity purification [email protected] www.abclonal.com.cn Storage Store at -20℃.