沈哲鯤臺灣大學:分子醫學研究所賈亞Krishnan, Bose JayaramaBose JayaramaKrishnan2010-05-042018-07-092010-05-042018-07-092008U0001-0710200809582000http://ntur.lib.ntu.edu.tw//handle/246246/178689TDP-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 multifunctionalNA-binding protein for a diverse set of cellular activities.Table of Contents 1bstract 3ntroduction 4xperimental rationale 18aterials and Methods 19lasmid Constructs 19xpression Vectors 20NA transfection and In vivo splicing assay 20NA immunoprecipitation (RNA-IP) 21rotein-protein interaction assay 22ligonucleotide preparation by in vitro transcription 23n vitro RNA-protein binding 24mmunostaining 25NA interference (RNAi) assay 25rimers Used 26esults 29DP-43 interacted with the SMN transcripts 29DP-43 stimulated the inclusion of exon 7 during splicing of SMN2 pre-mRNA 30DP-43 overexpression does not affect SMN1 splicing 31ifferential requirement of RRM domains for RNA binding and Splicing 31he AG-rich SE2 element of the SMN transcripts was required for TDP-43 stimulated exon 7-inclusion 33DP-43 binding with SMN-mRNA was not sequence specific 35DP-43 interacts with Htra2-β1 ......................... 36o-expression of TDP-43 and Htra2-β1................... 37NAi knockdown of TDP-43 had no effect on SMN splicing .. 38ransgenic mice ....................................... 39eferences .................................46igure....................................53ppendix ......................................70application/pdf2658715 bytesapplication/pdfen-USSMN基因SMN Exon-7http://ntur.lib.ntu.edu.tw/bitstream/246246/178689/1/ntu-97-D91448007-1.pdf