Mechanism of splicing

Transcription

1 Outline of Splicing

2 Mechanism of splicing

3 Em visualization of precursor-spliced mrna in an R loop

4 Kinetics of in vitro splicing

5 Analysis of the splice lariat

6 Lariat Branch Site

7 Splice site sequence requirements

8 Yeast Spliceosome

9 Human spliceosome

10 Alternative Splicing

11 Snurps or SnRNPs Small nuclear ribonuclear protein particles U1, U2, U4, U5 and U6 involved in nuclear RNA splicing Snurps constitute components of the spliceosome and are key components of splicing Each snurp has a unique role is splicing and the sequence of the RNA component of the snurp determines the role played in splicing

12 U1 interacts with the 5 splice junction: Mutations in the 5 Intron can be compensated by mutations in the U1 RNA

13 Mutations in the 5 intron can be compensated by mutations in the U1 RNA

14 The U6 snrnp also interacts with the 5 splice junction

15 U2 base pairs with the yeast branch-point sequences within the intron

16 Summary of the SnRNP s Roles in Splicing U1 binds the exon1-intron border U2 with the aid of U2AF binds the lariat junction sequence in the intron U6, U5 and U4 are a preassembled trimer which associate with U1 and U2 to bring the exons into alignment

17 Splicing and SnRNPs U6 associates with the 5 end ot the intron by base pairing U6 binds to U2 U5 associates with the last nucleotide of the exon1 and the first of exon2 U1 is released U4 is bound to U6 transiently until U6 is needed in the splicing reaction

18 Splicing and SnRNPs U4 is released and U5 binds stably to the 5 and 3 splice sites U2 and U6 catalyze the transesterification reaction forming the lariat and ligating the exon junctions

19 U6 and U5 Crosslinks to the pre-mrna

20 U2 U6 and U5 RNA interactions

21 The Spliceosome Cycle

22 3 Splice site selection Specific RNA binding proteins including the SR class of factors determine the selection of the 3 splice site

23 The yeast commitment complex A branchpoint bridging protein BBP binds to a protein in the U1 SnRNP at the 5 end of the intron and to Mud2p at the 3 end of the intron Thus these additional proteins may play a important initial role in setting up the spliceosome in yeast

24 The yeast commitment complex

25 Alternative Splicing: Immunoglobulin genes

26 Alternative Splicing: Sex determination in Drosophila

27 Group I Introns self splice Tetrahymena 26S rrna precursor intron can be removed in vitro with no protein addition to the RNA The reaction requires a guanine nucleotide on the 5 splice site, adding G to the 5 end of the intron and releasing the first exon The first exon attacks the 3 splice site ligating the two exons together and releasing the linear intron

28 Tetrahymena 26S rrna self splicing

29 What happens to the intron?

30 trna Splicing

31 Mechanism of trna splicing The intron is removed by a membrane bound endonuclease leaving a 2-3 phosphate group The 5 end of the 3 half of the molecule is phosphorylated by a kinase and the end is further modified by ligase to add an AMP molecule The two half-molecules are ligated through a 3-5 phosphodiester linkage Finally the 2 phosphate group is removed and a mature trna is formed

32 Mechanism of trna splicing

33 Precursor mrnas are capped Soon after polymerase clearance of the promoter a CTD bound enzyme guanylylmethyltransferase adds an inverted GDP molecule to the 5 end of the transcript

34 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

35 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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37 Functions of Caps Protect mrna from degradation Enhance translation of the mrna Enhance the nuclear export of the mrna Enhance the efficiency of splicing

38 Poly AMP addition to the 3 end of the mrna Poly (A) polymerase adds approximately 250 AMP residues to the transcript Poly (A) increases the lifetime of the mrna and its ability to be translated A specific recognition sequence AAUAAA occurs approximately 20 bases before the poly (A) site

39 Poly (A) consensus sequence

40 Cleavage and poly (A) addition

41 Figure 15.26

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