Proteomic analysis of the pre-mRNA splicing machinery utilizing chromosomal locus epitope tagging in metazoans

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Title: Proteomic analysis of the pre-mRNA splicing machinery utilizing chromosomal locus epitope tagging in metazoans
Author: Chen, Yen-I Grace, 1977-
Abstract: Epitope tagging in metazoans is an important tool for biochemical analyses and is generally accomplished by using trans -genes with in -frame epitope tags . However , protein levels from trans -genes are rarely representative of native levels . To overcome the shortcomings using trans -genes , epitope tags were introduced by homologous recombination technology , termed CLEP tagging (Chromosomal Locus EPitope tagging ) , immediately upstream of the stop codon of targeted genes in chicken B cell line DT40 and mouse embryonic stem (ES ) cells . I first demonstrated the feasibility and promise of this technique in DT40 cells by purifying low abundance polypeptides and factors loosely associated with the SmD3 protein , a core protein participating in pre -mRNA splicing and mRNA turnover , with a TAP (tandem affinity purification ) tag . Glycerol gradient separation was performed to further characterize the SmD3 -associated protein complexes from the 200S fractions , corresponding to the supraspliceosomes . The purification included all five spliceosomal snRNAs . Most known snRNP -associated proteins , 5' end binding factors , 3' end processing factors , mRNA export factors , hnRNPs , and other RNA binding proteins were identified from the protein components . Intriguingly , the purified supraspliceosomes also contained a number of structural proteins , nucleoporins , chromatin remodeling factors , and several novel proteins that were absent from splicing complexes assembled in vitro . I showed that the in vivo analyses provide a more comprehensive list of polypeptides associated with pre -mRNA splicing apparatus as well as those that coupled transcription to the pre -mRNA processing steps . With similar techniques , the TAP tag was inserted into the chromosomal locus of a pre -mRNA splicing factor component , mSART -1 in live mice . Surprisingly , a profound autoimmune response was induced in homozygous -modified mice , due likely to an inappropriate stimulation of the immune system . I believe these mice will serve as a valuable tool for the studies of mammalian autoimmune diseases , especially those resulting from the generation of autoantibodies against RNP components .
URI: http : / /hdl .handle .net /2152 /3213
Date: 2008-08-28

Citation

Proteomic analysis of the pre-mRNA splicing machinery utilizing chromosomal locus epitope tagging in metazoans. Doctoral dissertation, The University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /3213 .

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