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1 IMPACT of the POLY(A) LIMITING ELEMENT on MRNA 3' PROCESSING EFFICIENCY and TRANSLATION DISSERTATION Presented in Partial Fu

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1 IMPACT of the POLY(A) LIMITING ELEMENT on MRNA 3' PROCESSING EFFICIENCY and TRANSLATION DISSERTATION Presented in Partial Fu IMPACT OF THE POLY(A) LIMITING ELEMENT ON MRNA 3’ PROCESSING EFFICIENCY AND TRANSLATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Graduate School of The Ohio State University By Jing Peng, M.A. ∗∗∗∗∗ The Ohio State University 2004 Dissertation Committee: Approved by Professor Daniel R. Schoenberg, Advisor Professor Kathleen A. Boris-Lawrie Professor Lee F. Johnson Professor Michael C. Ostrowski Advisor Graduate Program in the Ohio State Biochemistry Program 1 ABSTRACT The poly(A)-limiting element (PLE) is a cis-acting sequence whose presence in the terminal exon results in the addition of a short, discrete <20 nt poly(A) tail on reporter mRNA. This study has examined the 3’ processing and translation efficiencies of PLE-containing mRNAs with short poly(A) tails. In cells transfected with the human β-globin reporter genes with or without a PLE, the PLE increases the accumulation of β-globin mRNA in both nuclear and cytoplasmic RNA fractions. Quantitative RT-PCR and RNase protection assays showed that the PLE increases pre-mRNA 3’ cleavage in vivo to the same degree that it increases the amount of β-globin mRNA. Moreover, in vitro cleavage assays also indicated that the PLE enhances 3’ processing efficiency. Thus, in addition to restricting the length of the poly(A) tail to <20 nt, the PLE also acts as an enhancer of pre-mRNA 3’ processing. A firefly luciferase reporter gene was used to examine the translation efficiency of PLE-containing mRNAs with short poly(A) tails. In transfected cells, PLE-containing mRNA with a <20 nt poly(A) tail associated with polysomes and was translated as well as the matching control mRNA with a long poly(A) tail. The impact of the PLE and poly(A) tail length on translation in vitro was examined using a cap- and poly(A)-dependent in vitro translation system ii prepared from HeLa cells. In vitro the presence of a PLE did not overcome the negative effect of a short poly(A) tail on translation. A similar result was observed for capped and polyadenylated mRNAs directly transfected into cells, suggesting that a nuclear processing event facilitates PLE-stimulated translation of short poly(A) mRNA. The addition of the PLE-binding proteins did not selectively enhance the translation of PLE-containing mRNAs with short poly(A) tails in vitro. However, selective PLE-enhanced translation of short poly(A) mRNA was observed in vitro when PABP activity was inhibited. Although the translation of PLE-containing mRNAs was still less efficient than that observed in vivo, these data indicate that the PLE functionally substitutes for bound PABP to stimulate translation of short poly(A) mRNA. iii Dedicated to all of the people who supported me through this work. iv ACKNOWLEDGMENTS I wish to express my appreciation to my advisor and mentor, Dr. Daniel R. Schoenberg, whose advice and encourage have been essential to my development as a scientist. He has taught me how to think critically and has given me motivation and support throughout my training. I would like to thank the members of my dissertation committee, Dr. Kathleen Boris-Lawrie, Dr. Lee Johnson, and Dr. Michael Ostrowski for their support, encouragement and stimulating discussions. I want to thank Dr. Nahum Sonenberg and Dr. Reinhard Lϋhrmann for providing reagents. I would like to thank my colleague Dr. Beth Murray for sacrificing her leisure time to edit this dissertation. I also want to thank all the Schoenberg Lab members who have educated and inspired me, including Joy, Rob, Venk, Changhong, Feng, Emily, Yan, Ravi, Yong, Jennifer, Noah, Yuichi, Merlin, and especially Kris, Mark, Haidong and Kirsten who have taught me so much. I would like to thank my husband Dong Xie, my mother Li Tang and father Xiaotang Peng for their unconditional love and support. I want to thank the Ohio State Biochemistry Program for its support and the NIH for funding. v VITA July 23, 1975 …………………………Born – Guiyang, P. R. China 1992 – 1996 ………………………… B.S. Microbiology, Nankai University 1996 – 1998 ………………………… M.A. Biochemistry, Temple University 1998 – present ……………………… Graduate Research Associate, The Ohio State University. FIELDS OF STUDY Major Field: Ohio State Biochemistry Program vi TABLE OF CONTENTS Page ABSTRACT……………………………………………………………………………...ii DEDICATION……………………………………………………………………….…..iv ACKNOWLEDGMENTS…………………………………………………………….….v VITA……………………………………………………………………………………...vi TABLE OF CONTENTS………………………………………………………………vii LIST OF TABLES………………………………………………………………..……xiii LIST OF FIGURES……………………………………………………………………xiv ABBREVIATIONS……………………………………………………………………xvi CHAPTERS: 1. INTRODUCTION………………………………………………..1 1.1 Formation of the poly(A) tail: 3’ end processing………....….2 1.1.1 Mammalian cleavage/polyadenylation…………………....…..3 1.1.1.1 Mammalian cleavage/polyadenylation signal…………..…….3 1.1.1.2 Mammalian cleavage/polyadenylation machinery…..…..…..6 1.1.1.2.1 Cleavage/polyadenylation specific factor (CPSF)……..….....6 1.1.1.2.2 Cleavage stimulation factor (CstF)………………………….…7 1.1.1.2.3 Cleavage factors Im and IIm (CF Im and CF IIm)……….…..….8 1.1.1.2.4 Poly(A) polymerase (PAP)…………………………………......8 1.1.1.2.5 Poly(A) binding protein N1 (PABPN 1)…………………….....9 1.1.1.2.6 The C-terminal domain of RNA polymerase II (CTD of RNAP II)………………………………………………………....9 1.1.1.3 The process of 3’ end formation in mammalian cells……...10 1.1.2 Yeast cleavage/polyadenylation………………………….….11 1.1.2.1 Yeast polyadenylation signal …………………………….…..11 1.1.2.2 Yeast cleavage/polyadenylation machinery………………...11 1.1.2.3 The process of 3’ end formation in yeast cells……………..15 vii 1.2 Poly(A) tail and translation initiation: circular translation model……………………………………………………….…..16 1.2.1 General pathway of translation initiation……………………17 1.2.2 Three key protein factors in the circular translation Model………………………………………………...……….…18 1.2.2.1 The cap binding protein: eIF4E……………………...……….18 1.2.2.2 The scaffold protein: eIF4G……………………………..……19 1.2.2.3 The Poly(A) binding protein: PABP.………..……….….……20 1.2.3 Circularization of translating mRNAs………………..………22 1.2.4 Translation of poly(A)-deficient histone mRNA………..……24 1.2.5 Translational control at the cap-eIF4E-eIF4G-PABP- poly(A) interface.…………………………………………….…26 1.2.5.1 eIF4E binding proteins (4E-BPs)…………………………….26 1.2.5.2 PABP interacting proteins (Paip)………………………….…26 1.2.5.3 Cytoplasmic polyadenylation mediated translational control…………………………………………………………..27 1.2.5.4 Viral infection…………………………………………………..28 1.2.5.4.1 Picornaviruses…………………………………………………29 1.2.5.4.2 Rotavirus………………………………………………………..30 1.2.5.4.3 Influenza virus………………………………………………….31 1.2.6 Length dependent translation stimulation by the poly(A) tail……………………………………………………………….32 1.3 Poly(A) limiting element (PLE)……………………………….32 1.3.1 Sequence of PLE…………………………..………………….34 1.3.2 PLE binding proteins: PLE-BPs………………………….…..34 1.4 Specific aims of this study…………………………………….36 1.4.1 Determine whether the PLE also functions to modulate the efficiency of pre-mRNA 3’ processing…………………..37 1.4.2 Examine impact of the PLE on the translation of mRNAs with short poly(A) tails….……………………………38 1.4.3 Analyze the effect of PLE-BPs and modulators of PABP on the translation of PLE-containing mRNAs with short poly(A) tails…………………………………………39 2. MATERIALS AND METHODS……………………………….46 2.1 Plasmid construction………………………………………….46 2.1.1 Plasmids used for transfection………………………………46 2.1.1.1 CMV-luc-SPA and CMV-luc-PLEB-SPA…………………….46 viii 2.1.1.2 CMV-glo-SPA-X200, CMV-glo-PLEB-SPA-X200 and CMV- glo-MutG-SPA-X200…………………………………………..47 2.1.1.3 pcDNARluc……………………………………………………..47 2.1.2 Plasmids used as templates to generate antisense probes……………………………………………………….….47 2.1.2.1 pTopo-gloA2……………………………………………….…...47 2.1.2.2 pBS-X200……………………………………………………….48 2.1.2.3 pGEMPL156……………………………………………………48 2.1.2.4 pGEMRL289…………………………………………………...48 2.1.2.5 pBSPL156 and pBSRL289…………………………….….….49 2.1.2.6 pTopII-Alb……………………………………………….….…..49 2.1.3 Plasmids used as templates to generate transcripts for in vitro cleavage assays……………………………………....49 2.1.3.1 pGgloEx3SPA and pGgloEx3PLEBSPA…………………….49 2.1.3.2 pGEM(PLEB)4 and pGEM(MutG)4………………………...…50 2.1.3.3 pGEx3(PLEB)4SPA and pGEx3(MutG)4SPA…………….....50 2.1.4 Plasmids used as templates to generate luciferase transcripts with poly(A) tails of different lengths……………50 2.1.4.1 Cloning of part of β-globin exon3 with A14, A54 or A78 into the pGEM3Z vector………………………………….…...50 2.1.4.2 Insertion of the entire β-globin exon3 into the pGEM A14, A54 or A78 vectors…………………………..….51 2.1.4.3 pGglo-A14, A54, A78……………………………………….…51 2.1.4.4 pGluc-A14, A54, A78 and pGluc-PLEB-A14, A54, A78.…..51 2.1.4.5 pGluc-PLEB-A98 and pGluc-A98……………………….……52 2.1.4.6 pGluc-A20 and pGluc-PLEB-A20……………………….……52 2.1.4.7 pGRluc-A78……………………………………………….……52 2.1.4.8 pGluc-EPPLE-A20, A98………………………………………53 2.2 Cell culture……………………………………………….……..53 2.3 Transfection of Cos7 cells……………………………….……53 2.4 Transfection of LM(tk-) cells………………………………….54 2.5 Total RNA extraction…………………………………….…….55 2.6 Extraction of cytoplasmic and nuclear RNA…………….…..56 2.7 Labeling of the 5’ end of DNA oligo-nucleotides with [γ-32P]-ATP……………………………………………….….….57 2.8 Quantitative RT-PCR to detect pre-mRNA………………….57 2.9 The poly(A) tail length assay…………………………………58 2.10 In vitro transcription……………………………………………59 2.10.1 Internally [32P] labeled antisense transcripts…………….….59 ix 2.10.2 Internally [32P] labeled transcripts bearing an m7GpppG cap………………………………………………………………60 2.10.3 Transcripts for in vitro translation experiments……………..61 2.11 Synthesis of random primed DNA probe……………………62 2.12 Synthesis of 32[P]-labeled U6 probe…………………………63 2.13 Northern blot……………………………………………………63 2.14 RNase Protection Assay (RPA)……………………………...64 2.15 Nuclear transcription run-on assay…………………….…….65 2.16 In vitro cleavage assay…………………………………….….66 2.17 Sucrose density gradient analysis……………………….…..67 2.18 Preparation of Hela cytoplasmic extract…………………….69 2.19 Purification of GST-Paip2………………………………….….69 2.20 Purification of GST-La(226-348)………………………….….71 2.21 Purification of recombinant hPABP-(His)6………………......71 2.22 In vitro translation………………………………………….…..72 2.23 Western blot……………………………………………………73 3.
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