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



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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.