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研究生:賈亞
研究生(外文):Bose Jayarama Krishnan
論文名稱:TDP-43大量表現對於包含SMN基因之Pre-mRNA的Exon-7剪接之增益
論文名稱(外文):TDP-43 Overexpression Enhances Exon-7 Inclusion during SMN Pre-mRNA Splicing
指導教授:沈哲鯤沈哲鯤引用關係
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:97
語文別:英文
論文頁數:73
中文關鍵詞:SMN基因
外文關鍵詞:SMN Exon-7
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TDP-43 is a highly conserved, 43 kDa RNA-binding protein implicated to play a role in transcription repression, nuclear organization, and alternative splicing. More recently, TDP-43 has been identified as the major disease protein of several neurodegenerative diseases including frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). For the splicing activity, TDP-43 has been shown to be mainly an exon-skipping promoter. In this study using the Survival of Motor Neuron (SMN) minigenes as the reporters in transfection assay, I show for the first time that TDP-43 could also act as an exon-inclusion factor. Furthermore, both RRM domains are required for the ability of TDP-43 to enhance the SMN2 exon 7-inclusion. Combined protein-immunoprecipitation (IP) and RNA-IP experiments also suggested that the exon-inclusion by TDP-43 might be mediated by multimeric complex(es) consisting of TDP-43 interacting with other splicing factors including Htra2-β1. My data further evidences TDP-43 as a multifunctional
RNA-binding protein for a diverse set of cellular activities.
Table of Contents 1
Abstract 3
Introduction 4
Experimental rationale 18
Materials and Methods 19
Plasmid Constructs 19
Expression Vectors 20
DNA transfection and In vivo splicing assay 20
RNA immunoprecipitation (RNA-IP) 21
Protein-protein interaction assay 22
Oligonucleotide preparation by in vitro transcription 23
In vitro RNA-protein binding 24
Immunostaining 25
RNA interference (RNAi) assay 25
Primers Used 26
Results 29
TDP-43 interacted with the SMN transcripts 29
TDP-43 stimulated the inclusion of exon 7 during splicing of SMN2 pre-mRNA 30
TDP-43 overexpression does not affect SMN1 splicing 31
Differential requirement of RRM domains for RNA binding and Splicing 31
The AG-rich SE2 element of the SMN transcripts was required for TDP-43 stimulated exon 7-inclusion 33
TDP-43 binding with SMN-mRNA was not sequence specific 35
TDP-43 interacts with Htra2-β1 ......................... 36
Co-expression of TDP-43 and Htra2-β1................... 37
RNAi knockdown of TDP-43 had no effect on SMN splicing .. 38
Transgenic mice ....................................... 39
References .................................46
Figure....................................53
Appendix ......................................70
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