Figure mouse globin mrna PRECURSOR RNA hybridized to cloned gene (genomic). mouse globin MATURE mrna hybridized to cloned gene (genomic).

Transcription

1 Splicing

2

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

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. 4

5 MAMMALLIAN EXON INTRON EXON AG GUAAGU YNCURAC-YnNAG G SPLICE SITE * 3 SPLICE SITE YEAST EXON INTRON EXON GUAUGU UACUAA-YAG SPLICE SITE * 3 SPLICE SITE If 2 nucleotides are given then the upper one is the most common.

6 Splicing is a 2 step trans-esterification 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 nucleotides upstream of the 3' splice site. OH Exon 1 pgu a agp Exon2 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. Exon 1-OH Exon 1-p-Exon 2 UG p A AGp Exon2 UG p A OH Figure 14.4

7 Structure of the lariat 5' most nucleotide of intron 3'-P-5' U 5' P 3' G 5' P 2' A 3'-P-5' 3' most nucleotide of intron A3'-P-5'-G-3'-OH 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 Rise & Fall suggested that the top band was an intermediate.

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. Very hard to see band 4% polyacrylamide gel 8M urea 10% poly- acrylamide gel 8 M urea 9 Discussed in Weaver 3rd ed. pg 439.

10 Thin layer chromatography to demonstrate that A is the branchpoint 10 TLC is thin layer chromatography. Tell them what it is. Figure 14.7 Weaver 4rd edition. pg 405.

11 4% polyacrylamide gel 8M urea

12 Is the branched nucleotide attached to the 5 end of the intron? RNase T1 cuts after guanylate residues. RNase T1 cuts only single stranded RNA. If you know what the sequence of the molecule is then you can predict the cutting pattern. Unspliced mrna gave a predicted band UCCCUUUUUUUCCACAGp. Some splicing intermediates gave a band that had anomolous eletrophoretic migration.

13 Snurps snrnps pronounced snurps small nuclear ribonucleoprotein particle The particle contains small nuclear RNAs = snrnas small nuclear ribonucleoproteins pg 407

14 5 splice site also called the donor site 3 splice site also called the acceptor site 3 splice site about 18 to 40 nucleotides down stream of the branch point. 3 splice site should be 18 to 40 nucleotides down stream of the branch point. (pg 448).

15 Branchpoint Yeast consensus: UACUAAC Mammallian: U 47 NC 63 U 53 R 72 A 91 C 47 15

16 Importance of the branchpoint Figure th edition 16

17 Multiple products identified by RNase protection. Mutate individual splice sites and demonstrate a role for base-pairing between splice sites and U1.

18 Figure th ed.

19 Figure th ed.

20 Figure th ed.

21 Figure th ed. U6 base pairs with the 5 end of the intron prior to lariat formation. U4 may act as a chaparone for U6 but does not appear to have a direct role in splicing. Base pairing is requried but is it sufficient? U6 also must pair with the 5 splice site therefore, some mutations in the 5 splice site may require changes in U1 and U6 in order for suppression to occur (page 431, Weaver 2nd edition).

22 Figure th ed. U6 base pairs with the 5 end of the intron prior to lariat formation. U4 may act as a chaparone for U6 but does not appear to have a direct role in splicing. Base pairing is requried but is it sufficient? U6 also must pair with the 5 splice site therefore, some mutations in the 5 splice site may require changes in U1 and U6 in order for suppression to occur (page 431, Weaver 2nd edition).

23 Figure th ed. U6 base pairs with the 5 end of the intron prior to lariat formation. U4 may act as a chaparone for U6 but does not appear to have a direct role in splicing. Clearly base pairing with U1 is not all that is required. Base pairing is requried but is it sufficient? U6 also must pair with the 5 splice site therefore, some mutations in the 5 splice site may require changes in U1 and U6 in order for suppression to occur (page 431, Weaver 2nd edition).

24 Recognition of mammalian premrna intron sequences by snrnps 24

25 Fig Spliceosome Cycle

26 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. 3 splice site should be 18 to 40 nucleotides down stream of the branch point. (pg 448). Parallel spliceosome AT-AC U5 interacts with the last nucleotide in exon 1 and the first nucleotide of exon 2.

27 Parallel spliceosome AT-AC U5 interacts with the last nucleotide in exon 1 and the first nucleotide of exon 2.

28 Group II self splicing

29 Figure 14.22

30

31 Group I introns Tetrahymena rrna U p A U p A G p U U p A U p A p G p A U p A G p U U-OH G p U p G OH G p A U p A p G p A G G p A U-OH U p U Degrade An example of an Autocatalytic exon Tetrahymena pre-rrna

32 SR proteins Serine (S) and Arginine (R) rich proteins that help to identify exons Involved in alternative splicing 32

33 RNAP II CTD Experiment: CTD-GST stimulates splicing in vitro. GST does not. CTD binds snrnps and splicing proteins. 33

34 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. 34

35 Figure This topic begins on page 427.

36

37 U2SNRP U2SNRP U1 SNRP U2AF SXL SXL U1 SNRP U2AF 37

38 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). 38

39 39

40 end