RNA Rearrangements in the Spliceosome -extensive U4/U6 interaction is replaced with a U2/U6 structure -U1 displaced at 5’ splice site by U6
RNA Rearrangment at 5’ Splice Site
Why Splicing?
Why Splicing? -ancient or new? -functional units (W. Gilbert) -diversity of function, evolution -alternative splicing
Exon Shuffling
Sequence Conservation Within Introns 5’ Splice Site Yeast Human
Sequence Conservation Within Introns Branch Region 3’ Splice Site Yeast Human
Drosophila Doublesex (dsx) RNA
Tissue and Developmental Regulation
Alternative Splicing and Proteome Complexity Dscam 115 exons 20 constitutive, 95 alternatively spliced 38,016 possible proteins
Mechanisms of Alternative Splicing -activation/repression -intron sequences: splice sites, pyrimidine tract, branch region -exon sequences (purine rich enhancers)
Recall: Selection of 5’ Splice Site and Branch
SR Proteins -S100 extract is deficient in pre-mRNA splicing -S100 + SR splices -SR proteins are phosphoproteins (SRPK kinase) -human SC35, 24 C terminal SR repeats
RNA Recognition Motif (RRM) PTB RRM-3 Sxl RRM-1
Activation -downstream activation of 5’ splice site -adenovirus E1A, also Tra/Tra2 Mol. Cell. Biol. (1999) 19, 7347
“Enhancers”: -stimulate 5’ or 3’ splice site usage vs
“Enhancers”: -stimulate 5’ or 3’ splice site usage vs
“Enhancers”: -within exons -proximal to splice sites -conserved
Science 297, 1007 (2002)
Repression -formation of RNA secondary structure (blocking site access); relieved by ATP dependent helicases -binding of trans factors
Drosophila sxl Gene
sxl Regulation of Drosophila splicing -sxl competition with U2AF on dsx (also polypyrimidine tract binding protein -PTB; neuronal specific events)