DOCSLIB.ORG

The Impact of Cytoplasmic Capping on Transcriptome Complexity Dissertation Presented in Partial Fulfillment of the Requirements

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Impact of Cytoplasmic Capping on Transcriptome Complexity Dissertation Presented in Partial Fulfillment of the Requirements The impact of cytoplasmic capping on transcriptome complexity Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Daniel E. del Valle-Morales, B.S. Graduate Program in Molecular, Cellular, and Developmental Biology The Ohio State University 2020 Dissertation Committee Daniel R. Schoenberg, Advisor Dawn S. Chandler Ralf Bundschuh Guramrit Singh Copyrighted by Daniel E. del Valle-Morales 2020 Abstract The 5’ cap is an essential modification of mRNAs that is needed for the functionality and lifespan of an mRNA. The cap is added almost immediately after the first nucleotide is transcribed, coordinated by RNGTT and RNMT-RAM bound to the C-terminal tail of RNA Pol II. This process of capping was thought to exclusively occur in the nucleus and loss of the cap was irreversible, leading to the rapid degradation of the mRNA. However, not all mRNAs share this fate. Over the last decade, the Schoenberg lab has characterized cytoplasmic capping, a process where the cap can be restored to previously decapped mRNAs. Cytoplasmic capping is catalyzed by a complex that consists of a cytoplasmic pool of both RNGTT and RNMT-RAM bound to the adapter protein NCK1 along with an unknown 5’ monophosphate kinase. mRNAs that undergo cytoplasmic capping can cycle from being in a decapped state to a recapped state as a way to fine tune gene expression, a process called cap homeostasis. The recapping targets were initially identified using a catalytically inactive and cytoplasmically restricted form of RNGTT termed K294A. Overexpression of K294A resulted in an accumulation of uncapped mRNAs in non-translating mRNPs. A major drawback of this approach was its reliance on detecting uncapped mRNAs, a potentially unstable population that can undergo partial degradation. An alternative tool to study recapping was developed using a catalytically inactive and cytoplasmically restricted form of RNMT termed ΔN-RNMT. ΔN- RNMT expression results in a decrease in the steady state levels of recapped mRNAs. A U2OS stable cell line expressing ΔN-RNMT was developed, and changes in the steady state levels of potential recapping genes were measured by RNA-Seq. 5’ terminal oligopyrimidine (TOP) mRNAs were identified as recapping targets, and I showed direct evidence of recapping i occurring at both the canonical site and downstream in the 5’UTR of eIF3D and eIF3K. Expression of ΔN-RNMT also results in a shift in the 3’UTR usage from proximal sites to distal sites. Uncapped mRNAs that accumulated with inhibition of cytoplasmic capping map to downstream CAGE tags (Kiss et al., 2015). If recapping were to occur at these sites, N- terminally truncated proteins could be translated. In collaboration with the Wysocki lab, we examined the relationship of cytoplasmic capping with N-terminally truncated proteins and showed that half of the downstream N-termini identified decrease when cytoplasmic capping is inhibited. These downstream N-terminal peptides correspond to RNA binding proteins, and the mapped downstream N-termini have a translation start site near the vicinity of the peptide. These studies expand the scope of cytoplasmic capping showing both direct evidence of recapping at canonical and downstream sites, and evidence of cytoplasmic capping expanding the proteome through synthesis of truncated proteins. ii Dedication I can write a thesis in itself for all the people I would like to dedicate this to, but I do want dedicate this to my undergraduate mentor Carlos Ruiz. iii Acknowledgements I first would like to give thanks to my collaborators for their contributions to this work particularly to Bernice Agana for her fantastic work. Thank you to the Center for RNA Biology, the Department of Biological Chemistry and Pharmacology, and the OSU Graduate School for supporting my work with funding and awards and for providing excellent facilities to research and to meet new scientists. Thanks to the NIH for providing the supplemental grant which funded the majority of my research. I would also like to acknowledge my committee members, Ralf, Guramrit, and Dawn, for their tough, yet fair critiques of my work. I would like to thank all of the past lab members; Chandrama, my first lab mentor; Dan Kiss for training me as an RNA biologist, Jackson for his friendship, Shan-Qing, and the various undergraduates Gabe, Andrew, and Mikaela. Thanks to Mike Kearse and his lab for that brief moment that we shared a lab bench. And to Wen Tan for his extensive lab discussions. And thank you, Daniel Schoenberg, for taking me in as an undergraduate during my first research internship. That experience cemented my path towards my Ph.D. and it was a blast to work for you. Gracias por tu ayuda! iv Vita 2011 …………………………………………………………….. First Bilingual Preparatory dd dd School, Aguadilla, Puerto Rico 2014 …………………………………………………………….. B.S. Natural Science, d University of Puerto Rico, d d d Aguadilla 2014 to present …………………………………………………. Graduate Research Associate, d d The Ohio State University Publications del Valle-Morales D., Trotman J., Bundschuh R., Schoenberg D.R. (2020) Inhibition of cytoplasmic cap methylation identifies 5’ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5’ends. Nucleic Acids Res., 48(7), 3806–3815 Fields of study Major Field: Molecular, Cellular, and Developmental Biology v Table of contents Abstract………………………………………………………………………………………….. i Dedication………………………………………………………………………………………. iii Acknowledgements …………………………………………………………………………….. iv Vita ………………………………………………………………………………………………v List of Tables …………………………………………………………………………………. viii List of Figures ………………………………………………………………………………….. ix Chapter 1. Introduction: Recapping of Cytoplasmic mRNAs…………………………………... 1 Abstract…………………………………………………………………………………………. 1 Introduction…………………………………………………………………………………….... 1 The 5’ cap and canonical capping……………………………………………………………... 3 Early Evidence of Cytoplasmic Capping ……………………………………………………... 5 The Cytoplasmic Capping Complex ………………………………………………………….. 6 Characteristics of Recapping Targets …………………………………………………………10 Capping downstream of canonical capping sites …………………………………………….. 13 Unanswered Questions ……………………………………………………………………….. 15 Chapter 2. Inhibition of cytoplasmic cap methylation identifies 5’ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5’ends……………………………… 17 Abstract……………………………………………………………………………………….. 17 Introduction …………………………………………………………………………………... 18 Materials and Methods ……………………………………………………………………….. 20 Results ………………………………………………………………………………………... 27 Discussion ……………………………………………………………………………………..45 Acknowledgements……………………………………………………………………………. 48 Chapter 3. mRNA recapping increases proteome complexity by enabling translation downstream of canonical 5’ends……………………………………………………………………………... 50 Abstract……………………………………………………………………………………… 50 Introduction……………………...……………………………………………………………. 51 vi Materials and Methods ……………………………………………………………………… 52 Results ………………………………………………………………………………………. 61 Discussion …………………………………………………………………………………... 71 Chapter 4. Identification of cytoplasmic capping sites in mRNAs……………………………. 75 Abstract……………………………………………………………………………………… 75 Introduction………………………………………………………………………………….. 75 Cap analysis of gene expression…………………………………………………………….. 78 Cap-SMART………………………………………………………………………………… 81 TeloPrime……………………………………………………………………………………. 85 ReCappable Seq……………………………………………………………………………... 90 Chapter 5. Future Work and concluding remarks……………………………………………... 95 References……………………………………………………………………………………... 99 Appendix List of primers…………………………………………………………………........ 105 vii List of Tables Table 1 PANTHER analysis on ΔN-RNMT downregulated genes ………………………….... 31 Table 2 PANTHER analysis on ΔN-RNMT upregulated genes ………………………............. 41 Table 3 List of primers used in chapter 2……………………………………………………....105 Table 4 List of primers used in chapter 3………………………………………………………106 viii List of Figure Figure 1 Structure of the 5’ cap and the enzymatic steps of nuclear capping ………………….. 2 Figure 2 The cytoplasmic capping complex……………………………………………………. 6 Figure 3 Identification of recapping targets through the inhibition of cytoplasmic capping…… 9 Figure 4 Recapping targets accumulate in non-translating RNPs when cytoplasmic capping is inhibited.........................................................................................................................................10 Figure 5 5’end procession of mRNAs as a possible origin of downstream recapping targets.....13 Figure 6 Validation of ΔN-RNMT cell line………………………….………………………… 26 Figure 7 Metagene analysis of QuantSeq data…………………………………………………. 28 Figure 8 Position of sequence tags and quantitative changes of RNAs after expression of ΔN- RNMT……………………………………………………………………………….…………. 29 Figure 9 Differential expression on TOP mRNAs with ΔN-RNMT induction……………….... 32 Figure 10 Western blot analysis of TOP genes………………………………………………..... 33 Figure 11 Cap-end analysis detects a decrease at the canonical and downstream capping sites of TOP mRNAs……………………………………………………………………………………. 35 Figure 12 Induction of ΔN-RNMT does not activate the cellular stress response……………... 40 Figure 13 3’UTR usage analysis reveals a shift from proximal to distal 3’UTR cleavage sites. 42 Figure 14 PABPN1 protein levels increase with ΔN-RNMT induction……………………….. 44 Figure 15 Inhibition of cytoplasmic capping shows a minor impact in the proteome………… 60 Figure 16 Approach for the enrichment of N-termini generated
Load more

Recommended publications
  • Transcriptional Profile Analysis of RPGRORF15 Frameshift Mutation Identifies Novel Genes Associated with Retinal Degeneration
    Retinal Cell Biology Transcriptional Profile Analysis of RPGRORF15 Frameshift Mutation Identifies Novel Genes Associated with Retinal Degeneration Sem Genini,1 Barbara Zangerl,1 Julianna Slavik,1 Gregory M. Acland,2 William A. Beltran,1 and Gustavo D. Aguirre1 PURPOSE. To identify genes and molecular mechanisms associ- he term retinitis pigmentosa (RP) refers to a group of many ated with photoreceptor degeneration in a canine model of Tdifferent inherited retinal diseases characterized by pro- XLRP caused by an RPGR exon ORF15 microdeletion. gressive rod or rod–cone photoreceptor degeneration that causes subsequent visual impairment and blindness. Some of METHODS. Expression profiles of mutant and normal retinas were compared by using canine retinal custom cDNA microar- the causative genes have clear, well-identified roles (e.g., in- volvement in phototransduction, in maintaining photoreceptor rays. qRT-PCR, Western blot analysis, and immunohistochem- structure, or in RPE retinoid metabolism; RetNet: http://www. istry (IHC) were applied to selected genes, to confirm and sph.uth.tmc.edu/RetNet/ provided in the public domain by the expand the microarray results. University of Texas Houston Health Science Center, Houston, RESULTS. At 7 and 16 weeks, respectively, 56 and 18 tran- TX). However, there remain a large number of diseases caused scripts were downregulated in the mutant retinas, but none by genes with poorly understood functions and for which the were differentially expressed (DE) at both ages, suggesting mechanism linking the genes and/or mutations with photore- the involvement of temporally distinct pathways. Down- ceptor disease and degeneration is unknown. regulated genes included the known retina-relevant genes Among these is the RP3 form of X-linked RP (XLRP), a PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at uniformly severe, early-onset retinal disease in humans that is 16 weeks.
    [Show full text]
  • Supplementary Table S1. Upregulated Genes Differentially
    Supplementary Table S1. Upregulated genes differentially expressed in athletes (p < 0.05 and 1.3-fold change) Gene Symbol p Value Fold Change 221051_s_at NMRK2 0.01 2.38 236518_at CCDC183 0.00 2.05 218804_at ANO1 0.00 2.05 234675_x_at 0.01 2.02 207076_s_at ASS1 0.00 1.85 209135_at ASPH 0.02 1.81 228434_at BTNL9 0.03 1.81 229985_at BTNL9 0.01 1.79 215795_at MYH7B 0.01 1.78 217979_at TSPAN13 0.01 1.77 230992_at BTNL9 0.01 1.75 226884_at LRRN1 0.03 1.74 220039_s_at CDKAL1 0.01 1.73 236520_at 0.02 1.72 219895_at TMEM255A 0.04 1.72 201030_x_at LDHB 0.00 1.69 233824_at 0.00 1.69 232257_s_at 0.05 1.67 236359_at SCN4B 0.04 1.64 242868_at 0.00 1.63 1557286_at 0.01 1.63 202780_at OXCT1 0.01 1.63 1556542_a_at 0.04 1.63 209992_at PFKFB2 0.04 1.63 205247_at NOTCH4 0.01 1.62 1554182_at TRIM73///TRIM74 0.00 1.61 232892_at MIR1-1HG 0.02 1.61 204726_at CDH13 0.01 1.6 1561167_at 0.01 1.6 1565821_at 0.01 1.6 210169_at SEC14L5 0.01 1.6 236963_at 0.02 1.6 1552880_at SEC16B 0.02 1.6 235228_at CCDC85A 0.02 1.6 1568623_a_at SLC35E4 0.00 1.59 204844_at ENPEP 0.00 1.59 1552256_a_at SCARB1 0.02 1.59 1557283_a_at ZNF519 0.02 1.59 1557293_at LINC00969 0.03 1.59 231644_at 0.01 1.58 228115_at GAREM1 0.01 1.58 223687_s_at LY6K 0.02 1.58 231779_at IRAK2 0.03 1.58 243332_at LOC105379610 0.04 1.58 232118_at 0.01 1.57 203423_at RBP1 0.02 1.57 AMY1A///AMY1B///AMY1C///AMY2A///AMY2B// 208498_s_at 0.03 1.57 /AMYP1 237154_at LOC101930114 0.00 1.56 1559691_at 0.01 1.56 243481_at RHOJ 0.03 1.56 238834_at MYLK3 0.01 1.55 213438_at NFASC 0.02 1.55 242290_at TACC1 0.04 1.55 ANKRD20A1///ANKRD20A12P///ANKRD20A2///
    [Show full text]
  • RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases
    G C A T T A C G G C A T genes Review RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases Amber Willbanks, Shaun Wood and Jason X. Cheng * Department of Pathology, Hematopathology Section, University of Chicago, Chicago, IL 60637, USA; [email protected] (A.W.); [email protected] (S.W.) * Correspondence: [email protected] Abstract: Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases. Keywords: 5’ cap (5’ cap); 7-methylguanosine (m7G); R-loops; N6-methyladenosine (m6A); RNA editing; A-to-I; C-to-U; 2’-O-methylation (Nm); 5-methylcytosine (m5C); NOL1/NOP2/sun domain Citation: Willbanks, A.; Wood, S.; (NSUN); MYC Cheng, J.X. RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases. Genes 2021, 12, 627.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Eef2k) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy
    International Journal of Molecular Sciences Review Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy Bin Zhang 1 , Jiamei Zou 1, Qiting Zhang 2, Ze Wang 1, Ning Wang 2,* , Shan He 1 , Yufen Zhao 2 and C. Benjamin Naman 1,* 1 Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315800, China; [email protected] (B.Z.); [email protected] (J.Z.); [email protected] (Z.W.); [email protected] (S.H.) 2 Institute of Drug Discovery Technology, Ningbo University, Ningbo 315211, China; [email protected] (Q.Z.); [email protected] (Y.Z.) * Correspondence: [email protected] (N.W.); [email protected] (C.B.N.) Abstract: Eukaryotic elongation factor 2 kinase (eEF2K or Ca2+/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The Citation: Zhang, B.; Zou, J.; Zhang, expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in Q.; Wang, Z.; Wang, N.; He, S.; Zhao, many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient Y.; Naman, C.B.
    [Show full text]
  • The MAP4K4-STRIPAK Complex Promotes Growth and Tissue Invasion In
    bioRxiv preprint doi: https://doi.org/10.1101/2021.05.07.442906; this version posted May 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 The MAP4K4-STRIPAK complex promotes growth and tissue invasion in 2 medulloblastoma 3 Jessica Migliavacca1, Buket Züllig1, Charles Capdeville1, Michael Grotzer2 and Martin Baumgartner1,* 4 1 Division of Oncology, Children’s Research Center, University Children’s Hospital Zürich, Zürich, 5 Switzerland 6 2 Division of Oncology, University Children’s Hospital Zürich, Zürich, Switzerland 7 8 *e-mail: [email protected] 9 10 Abstract 11 Proliferation and motility are mutually exclusive biological processes associated with cancer that depend 12 on precise control of upstream signaling pathways with overlapping functionalities. We find that STRN3 13 and STRN4 scaffold subunits of the STRIPAK complex interact with MAP4K4 for pathway regulation in 14 medulloblastoma. Disruption of the MAP4K4-STRIPAK complex impairs growth factor-induced 15 migration and tissue invasion and stalls YAP/TAZ target gene expression and oncogenic growth. The 16 migration promoting functions of the MAP4K4-STRIPAK complex involve the activation of novel PKCs 17 and the phosphorylation of the membrane targeting S157 residue of VASP through MAP4K4. The anti- 18 proliferative effect of complex disruption is associated with reduced YAP/TAZ target gene expression 19 and results in repressed tumor growth in the brain tissue. This dichotomous functionality of the STRIPAK 20 complex in migration and proliferation control acts through MAP4K4 regulation in tumor cells and 21 provides relevant mechanistic insights into novel tumorigenic functions of the STRIPAK complex in 22 medulloblastoma.
    [Show full text]
  • Molecular Effects of Isoflavone Supplementation Human Intervention Studies and Quantitative Models for Risk Assessment
    Molecular effects of isoflavone supplementation Human intervention studies and quantitative models for risk assessment Vera van der Velpen Thesis committee Promotors Prof. Dr Pieter van ‘t Veer Professor of Nutritional Epidemiology Wageningen University Prof. Dr Evert G. Schouten Emeritus Professor of Epidemiology and Prevention Wageningen University Co-promotors Dr Anouk Geelen Assistant professor, Division of Human Nutrition Wageningen University Dr Lydia A. Afman Assistant professor, Division of Human Nutrition Wageningen University Other members Prof. Dr Jaap Keijer, Wageningen University Dr Hubert P.J.M. Noteborn, Netherlands Food en Consumer Product Safety Authority Prof. Dr Yvonne T. van der Schouw, UMC Utrecht Dr Wendy L. Hall, King’s College London This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Molecular effects of isoflavone supplementation Human intervention studies and quantitative models for risk assessment Vera van der Velpen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 20 June 2014 at 13.30 p.m. in the Aula. Vera van der Velpen Molecular effects of isoflavone supplementation: Human intervention studies and quantitative models for risk assessment 154 pages PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in Dutch and English ISBN: 978-94-6173-952-0 ABSTRact Background: Risk assessment can potentially be improved by closely linked experiments in the disciplines of epidemiology and toxicology.
    [Show full text]
  • Supplementary Information
    SUPPLEMENTARY INFORMATION Myeloperoxidase-derived 2-chlorohexadecanal is generated in mouse heart during endotoxemia and induces modification of distinct cardiomyocyte protein subsets in vitro Jürgen Prasch, Eva Bernhart, Helga Reicher, Manfred Kollroser, Gerald N. Rechberger, Chintan N. Koyani, Christopher Trummer, Lavinia Rech, Peter P. Rainer, Astrid Hammer, Ernst Malle, Wolfgang Sattler Table S1: Biological process gene ontology (GO) enrichment analysis. #term ID term description observed background false discovery matching proteins in network (labels) gene count gene count rate GO:0006457 protein folding 10 153 5.21e-09 Cct3,Cct5,Cct8,Fkbp4,Hsp90aa1,Hsp a1l,Hspb1,Pdia3,Pdia6,Tcp1 GO:0007339 binding of sperm to 6 36 4.02e-07 Aldoa,Cct3,Cct5,Cct8,Hspa1l,Tcp1 zona pellucida GO:0061077 chaperone-mediated 6 60 2.67e-06 Cct3,Cct5,Cct8,Fkbp4,Hspb1,Tcp1 protein folding GO:0017144 drug metabolic process 11 494 4.06e-06 Aldh2,Aldoa,Eno1,Gapdh,Hsp90aa1,I dh3a,Ldha,Ndufs2,Pgam1,Phgdh,Uq crc1 GO:2000573 positive regulation of 6 69 4.16e-06 Cct3,Cct5,Cct8,Ddx39b,Hsp90aa1,Tc DNA biosynthetic p1 process GO:0009987 cellular process 47 12459 4.22e-06 Alad,Alb,Aldh2,Aldoa,Cct3,Cct5,Cct8, Dctn2,Ddx39,Ddx39b,Des,Eef1g,Eef 2,Eif3f,Eif4a2,Eno1,Fdps,Fkbp4,Gap dh,Hnrnpl,Hsp90aa1,Hspa1l,Hspb1,I dh3a,Ldha,Lmna,Lyz1,Ndufs2,Pcna, Pdia3,Pdia6,Pgam1,Phgdh,Prph,Psm d13,Rpsa,Ruvbl2,Tcp1,Tuba3b,Tubal 3,Tubb3,Tubb6,Uap1l1,Uqcrc1,Uqcrc 2,Vim,Ywhab GO:1904851 positive regulation of 4 10 4.22e-06 Cct3,Cct5,Cct8,Tcp1 establishment of protein localization to telomere GO:0046031
    [Show full text]
  • Associated 16P11.2 Deletion in Drosophila Melanogaster
    ARTICLE DOI: 10.1038/s41467-018-04882-6 OPEN Pervasive genetic interactions modulate neurodevelopmental defects of the autism- associated 16p11.2 deletion in Drosophila melanogaster Janani Iyer1, Mayanglambam Dhruba Singh1, Matthew Jensen1,2, Payal Patel 1, Lucilla Pizzo1, Emily Huber1, Haley Koerselman3, Alexis T. Weiner 1, Paola Lepanto4, Komal Vadodaria1, Alexis Kubina1, Qingyu Wang 1,2, Abigail Talbert1, Sneha Yennawar1, Jose Badano 4, J. Robert Manak3,5, Melissa M. Rolls1, Arjun Krishnan6,7 & 1234567890():,; Santhosh Girirajan 1,2,8 As opposed to syndromic CNVs caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. Here, we propose a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, we identify a range of neurodevelopmental phenotypes for knockdown of indi- vidual 16p11.2 homologs in different tissues. We test 565 pairwise knockdowns in the developing eye, and identify 24 interactions between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppress or enhance cell proliferation phenotypes compared to one-hit knockdowns. These interac- tions within cell proliferation pathways are also enriched in a human brain-specific network, providing translational relevance in humans. Our study indicates a role for pervasive genetic interactions within CNVs towards cellular and developmental phenotypes. 1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA. 2 Bioinformatics and Genomics Program, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
    [Show full text]
  • Molecular Pharmacology of Cancer Therapy in Human Colorectal Cancer by Gene Expression Profiling1,2
    [CANCER RESEARCH 63, 6855–6863, October 15, 2003] Molecular Pharmacology of Cancer Therapy in Human Colorectal Cancer by Gene Expression Profiling1,2 Paul A. Clarke,3 Mark L. George, Sandra Easdale, David Cunningham, R. Ian Swift, Mark E. Hill, Diana M. Tait, and Paul Workman Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG [P. A. C., M. L. G., S. E., P. W.]; Department of Gastrointestinal Oncology, Royal Marsden Hospital, Sutton, Surrey [D. C., M. E. H., D. M. T.]; and Department of Surgery, Mayday Hospital, Croydon, Surrey [M. L. G., R. I. S.], United Kingdom ABSTRACT ment with a single dose of MMC4 and during a continuous infusion of 5FU. In this study, we report for the first time gene expression Global gene expression profiling has potential for elucidating the com- profiling in cancer patients before, and critically, during the period of plex cellular effects and mechanisms of action of novel targeted anticancer exposure to chemotherapy. We have demonstrated that the approach agents or existing chemotherapeutics for which the precise molecular is feasible, and we have detected a novel molecular response that mechanism of action may be unclear. In this study, decreased expression would not have been predicted from in vitro studies and that would of genes required for RNA and protein synthesis, and for metabolism were have otherwise been missed by conventional approaches. The results detected in rectal cancer biopsies taken from patients during a 5-fluorou- also suggest a possible new therapeutic approach. Overall our obser- racil infusion. Our observations demonstrate that this approach is feasible and can detect responses that may have otherwise been missed by con- vations suggest that gene expression profiling in response to treatment ventional methods.
    [Show full text]
  • The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells
    cells Article The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells Ella Alkalay, Chen Gam Ze Letova Refael, Irit Shoval, Noa Kinor, Ronit Sarid and Yaron Shav-Tal * The Mina & Everard Goodman Faculty of Life Sciences and The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 5290002, Israel; [email protected] (E.A.); [email protected] (C.G.Z.L.R.); [email protected] (I.S.); [email protected] (N.K.); [email protected] (R.S.) * Correspondence: [email protected] Received: 14 August 2020; Accepted: 21 August 2020; Published: 25 August 2020 Abstract: RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to determine the fate of nucleoplasmic RNA-binding proteins and nuclear speckles during the lytic cycle of the Kaposi’s sarcoma associated herpesvirus (KSHV). We found that nuclear speckles became fewer and dramatically larger, localizing at the nuclear periphery, adjacent to the marginalized chromatin. Enlarged nuclear speckles contained splicing factors, whereas other proteins were nucleoplasmically dispersed. Polyadenylated RNA, typically found in nuclear speckles under regular conditions, was also found in foci separated from nuclear speckles in infected cells. Poly(A) foci did not contain lncRNAs known to colocalize with nuclear speckles but contained the poly(A)-binding protein PABPN1. Examination of the localization of spliced viral RNAs revealed that some spliced transcripts could be detected within the nuclear speckles.
    [Show full text]
  • The Nuclear Poly(A) Binding Protein of Mammals, but Not of Fission Yeast, Participates in Mrna Polyadenylation
    Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT The nuclear poly(A) binding protein of mammals, but not of fission yeast, participates in mRNA polyadenylation UWE KÜHN, JULIANE BUSCHMANN, and ELMAR WAHLE Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany ABSTRACT The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the S. pombe ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of ∼250 nucleotides, as predicted from biochemical data. We have also purified S. pombe Pab2 and the S. pombe poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not S. pombe Pab2 in the polyadenylation of mRNA precursors. Keywords: poly(A) binding protein; poly(A) polymerase; mRNA polyadenylation; pre-mRNA 3′; processing INTRODUCTION by poly(A) polymerase with the help of the cleavage and poly- adenylation specificity factor (CPSF), which binds the polya- The poly(A) tails of eukaryotic mRNAs are covered by specif- denylation signal AAUAAA.
    [Show full text]