EUKARYOTICGENEEXPRESSION-Enzimológiai…真核基因表达enzimoló解放….pptVIP

Discovery of Alternative Splicing - Alternative splicing gives two forms of the protein with different C-termini: - 1 form is shorter and secreted - Other stays anchored in the plasma membrane via C-terminus Splicing the spliceosome Structure 60S dynamic structure – a large complex consisting of ~ 150 proteins Five small nuclear RNAs (U1, U2, U4, U5 U6) RNAs assemble with proteins to form snRNPs (“snurps”) Protein splicing factors Assembly of spliceosome requires ATP Splicing defects Estimation: 15% of all genetic diseases associated with mutated splice sites snRNAs U1 snRNA Contains conserved sequence complementary to 5’ splice site of nuclear mRNA introns Contains pseudouridine (y) Splice-site recognition Splice Site Conservation Splice Site Conservation Splicing mechanism How Prevalent is Alternative Splicing? Regulation of/by Alternative Splicing Sex determination in Drosophila involves 3 regulatory genes that are differentially spliced in females versus males; 2 of them affect alternative splicing 1. Sxl (sex-lethal) - promotes alternative splicing of tra (exon 2 is skipped) and of its own (exon 3 is skipped) pre-mRNA 2. Tra – promotes alternative splicing of dsx (last 2 exons are excluded) 3. Dsx (double-sex) - Alternatively spliced form of dsx needed to maintain female state Nonsense Mediated Decay Evolutionary Overview of Alternative Splicing Introns unlikely to have been derived from ancient genes Multi-intron genes probably predated alternative splicing Most eukaryotes have introns but alternative splicing prevalent only in multicellular organism S.cerevisiae has only 253 introns (3% of its genes) and only 6 genes have 2 introns S. pombe: 43% of its genes have introns (usually 40-75 nt) S.cerevisiae and S. pombe have NO alternative splicing Compare cDNA genomic DNA sequences Compare ESTs genomic DNA sequences Compare protein genomic sequences Large-scale multiple alignment of expressed sequences Databases: tens of thousands of