1. RNA Rearrangements in the Spliceosome
-extensive U4/U6 interaction is replaced
with a U2/U6 structure
-U1 displaced at 5’ splice site by U6

2. RNA Rearrangment at 5’ Splice Site

3. Why Splicing?

4. Why Splicing? -ancient or new? -functional units (W. Gilbert)
-diversity of function, evolution
-alternative splicing

5. Exon Shuffling

6. Sequence Conservation Within Introns
5’ Splice Site
Yeast
Human

7. Sequence Conservation Within Introns
Branch Region
3’ Splice Site
Yeast
Human

8. Drosophila Doublesex (dsx) RNA

9. Tissue and Developmental Regulation

10. Alternative Splicing and Proteome Complexity
Dscam
115 exons
20 constitutive, 95 alternatively spliced
38,016 possible proteins

11. Mechanisms of Alternative Splicing
-activation/repression
-intron sequences: splice sites, pyrimidine tract,
branch region
-exon sequences (purine rich enhancers)

12. Recall:
Selection of 5’ Splice Site and Branch

13. 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

14. RNA Recognition Motif (RRM)
PTB RRM-3
Sxl RRM-1

15. Activation -downstream activation of 5’ splice site
-adenovirus E1A, also Tra/Tra2
Mol. Cell. Biol. (1999) 19, 7347

16. “Enhancers”:
-stimulate 5’ or 3’ splice site usage vs

17. “Enhancers”:
-stimulate 5’ or 3’ splice site usage vs

18. “Enhancers”:
-within exons
-proximal to splice sites
-conserved

19. Science 297, 1007 (2002)

21. Repression
-formation of RNA secondary structure (blocking site access); relieved
by ATP dependent helicases
-binding of trans factors

22. Drosophila sxl Gene

23. sxl Regulation of Drosophila splicing
-sxl competition with U2AF on dsx (also polypyrimidine tract binding protein
-PTB; neuronal specific events)