1. Splicing

2. Movie removed to simplify downloading. You can download the movie separately.

3. Figure 14.3 mouse globin mRNA PRECURSOR RNA hybridized to cloned gene (genomic). mouse globin MATURE mRNA hybridized to cloned gene (genomic).

4. 4 mRNA Splicing rRNA and tRNA are also sometimes spliced; however, these are not the current topic. Splice sites are strongly conserved Shapiro and Senapathy examined 3700 splice sites. Immediately before 3' splice site is pyridine rich and free of AG dinucleotide. Invariant consensus is GU at 5' donor site and AG at 3' acceptor site. This is usually referred to as the GT/AG rule.

5. MAMMALLIAN EXON |-------INTRON-------------------| EXON 5’---------AG GUAAGU-------------YNCURAC-YnNAG G---------3’ | | | 5’ SPLICE SITE * 3’ SPLICE SITE YEAST EXON |-------INTRON-------------------| EXON 5’----------- GUAUGU----------------UACUAA-YAG ---------3’ | | | 5’ SPLICE SITE * 3’ SPLICE SITE

6. Splicing is a 2 step trans-esterification Figure 14.4 Transesterification 1. First the 5' transesterification occurs and it generates 2'-5' phosphodiester bond. The 2' attachment point is a 2' hydroxyl of an A nucleotide within the intron. This position is called the branch point. The branch point is 17 - 40 nucleotides upstream of the 3' splice site. Transesterification 2. Second to occur is the 3' splice site cleavage, with simultaneous exon ligation. Intron leaves the complex as a lariat. The number of phophodiesters is conserved. No energy is lost or consumed.

7. Notice the 2' to 5' phosphodiestion bond at the branch point. Spliceosome does splicing This is a very large macromolecular complex. Spliceosomes are about 25 nm X 50 nm. It assembles on the mRNA. Assembly consumes ATP.

8. Weaver page 404

9. 9 Sharp & coworkers L1----IVS----L2 intron is 231n DO is an antiSNURP sera ME is the control sera Panel C is a Southern blot of the 10% acrylamide 8M urea gel probed with a L1L2 fragment. 4% poly- acrylamide gel 8M urea 10% poly- acrylamide gel 8 M urea Very hard to see band

10. 10 Thin layer chromatography to demonstrate that A is the branchpoint

11. RNase T1 cuts after guanylate residues. RNase T1 cuts only single stranded RNA. Is the branched nucleotide attached to the 5’ end of the intron?

14. Snurps snRNPs pronounced snurps –small nuclear ribonucleoprotein particle The particle contains –small nuclear RNAs = snRNAs –small nuclear ribonucleoproteins pg 407

15. 5’ splice site also called the donor site 3’ splice site also called the acceptor site

16. 16 Branchpoint Yeast consensus: UACUAAC Mammallian: U 47 NC 63 U 53 R 72 A 91 C 47 16

17. 17 Importance of the branchpoint Figure 14.8 4th edition

19. Figure 14.12

22. Base pairing is requried but is it sufficient?

24. Clearly base pairing with U1 is not all that is required.

25. 25 Recognition of mammalian pre- mRNA intron sequences by snRNPs

26. Spliceosome Cycle Fig 14.28

27. 27 SR proteins Serine (S) and Arginine (R) rich proteins that help to identify exons Involved in alternative splicing 27

28. snRNAs U1 - recognition (bp) of the 5’ splice site (donor site). U2 - branch point recognition (bp) & bps to U6 snRNA U5 binds ends of exons U4 binds U6 and holds it untile U6 is needed in a splicing reaction U6 - bps to 5’ splice site and U2 snRNA & U4 snRNA 3’ splice site should be 18- 40n downstream of branch point. Slu7 and U2AF use branch point to help recognize 3’ splice site.

30. Group II self splicing

31. Self splice and non-self splicing Group II mitochondria

32. Figure 14.22

33. Group I introns rRNA

34. 34 RNAP II CTD Experiment: CTD-GST stimulates splicing in vitro. GST does not. CTD binds snRNPs and splicing proteins. 34

35. 35 Exon definition - intron definition Intron definition is sufficient to identify ends of introns. –For some transcripts the splicing machinery identifies the ends of introns without help from CTD. Exon definition is needed to successfully identify the ends of introns –Here CTD helps to identify the ends of the EXONS. –These types of transcripts are not spliced if the exons are not whole. 35

36. Figure 14.37. This topic begins on page 427.

39. 39 U1 SNRP U1 SNRP U2SNRP U2AF U2SNRP U2AF SXL

40. 40 Regulation of splicing Negative - SR protein binds and hides a required sequence Positive - a crummy sequence (eg branch point) is enhanced with the help of a protein (eg U2AF). 40

41. 41

42. end