RNA Splicing

1. RNA Splicing • Prokaryotic genes: the codon is immediately adjacent to another • Eukaryotic genes: consists of blocks of coding sequences separated by noncoding sequences • Thus! RNA splicing is necessary to eliminated these noncoding sequences.

2. outline • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

3. RNA splicing: the process of removal of introns from the pre-mRNA. • * the significance of precision of splicing : since the translation is conducted in a fixed frame, even one base lost or insertion would result in the change of the reading frame, which would further result in error protein…

4. The chemistry of RNA splicing • 1. structural foundation A. 3’ splice site: B. 5’ splice site C. branch point site

5. 2. Process of reaction 2’ OH of A at branch site as nucelophile to attack the phosphoryl group of G in the 5’ splice site phosphodiester bond cleavage between intron and exon at 5’ splice site

6. Freed 5’ end joined to the A within the branch site • 5’ exon as a new nucleophile to attack the phosphoryl group at 3’ splice site 5’ exon and 3’ exon are spliced ; intron lariat is released

7. Consequence: two phosphodiester bonds formation at the cost of another two phosphodiester bonds equivalent bargain! • However , large amount of ATP required . For what? Splicing machinary

8. outline • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

9. The spliceosome machinery Process : U1 attached to 5’ splice site BBP binds to A within branch site U2AF binds to Py tract U2 binds to branch site aided by U2AF to replace BBP A residue extruded as bulge resulting form the base pair between U2 and branch site

10. Tri-snRNP particle: U6 U4 U5 recruited to join in the complex • U2AF released • U6 binds to 5’ splice site replacing U1

11. U4 released, allowing interactions between U6 and U2 to form active site • First transesterification • Second transesterification then two exons linked

12. Self-splicing introns • Definition : the intron itself folds into specific conformation within the precursor RNA and catalyze the chemistry of its own release . • without the involvement of other RNA or protein • 1. Group I Intro 2. Group II Intron

13. Group I intron release a linear intron rather than a lariat • Essential difference • free G nucleotide • Nucleophile used • A residue branch site

14. Group I self-splicing • Internal guide sequence base-pairs with 5’ splice site determining precisely which site for nucleophile attack by the G nucletide

15. Compares • Represents tendency of evolution • For self-splicing, intron sequences are restricted according to the need of formation of specific configration for self-splicing • For pre-mRNA spliceosome, the requirment above of intron sequence is released with the help of spliceosome

16. Three class of RNA Splicing

18. How does the spliceosome find the splice sites reliably? • Splice-site recognition is prone to errors • 1. splice site could be skipped • 2.close sequence but not legitimate splice sites mistakenly recognized .pseudo splice site

19. Guard against inappropriate splicing • 1.active site formation only formed on RNA sequences that pass the testing of being recognized by multiple elements during spliceosome assembly • 2.co-transcriptional loading process: correct 3’ splice site could be recognized before the formation of competing sequence downstreams

20. 3. ESE(exonic splicing enhancers): SR proteins bind to ESE and then recruit component of spliceosome(U2AF&U1) around the ESE.

21. outline • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

22. Alternative splicing • Single genes could produce multiple products by alternative splicing • Constitutive: more than one product is always made • regulated :different forms are generated at different times under different conditions

23. Constitutive splicing of T antigen genes

24. Alternative splicing is regulated by activators and repressors • Activator : • directing the splice machinery to related exons to activate splicing • Repressor: • inability to recruit the splice machinery but blocking splice site to prevent splicing

26. One example for activator • SR protein • One domain (RNA-recognizing motif) for binding to RNA • Another domain (RS domain ) to interact with splicing machinery • Recognizing the splice site with RRM and then use RS domain to recruit splice machinery to activate splicing

27. One example for repressor • hnRNP (heterogeneous nuclear ribonucleoprotein) • hnRNPA 1 binds to an exonic silencer • hnRNPA promote cooperative binding of additional molecule to cover the enhancer site • SC35(SR) fails to activate splicing

28. Another mammalian splicing repressor • hnRNP I • Through binding to Py tract • Consequence:exclude one exon from mature RNA • Reasons 1. loop out the exon • 2. cooperative molecule to cover • the whole exon • Both render the exon “invisible “to splice machinery!

29. The consequence of alternative splicing • 1. generation of diverse proteins( isoforms) form only one gene. 2. regulating the use of intron, which, if retained in the mRNA, the latter would never be transported outside the nucleus thus would never be translated.

30. outline • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

31. Exon shuffling • Why would introns only presents in eukaryotes but not in bacterias??? • Two theory • 1.intron early model • introns did exist in bacteria but are excised to the need of the increasing the rate of replication and cell division • 2.intron late moder • Introns are inserted later in eukaryotes by transposon-like mechanism.

32. Evidence for exon shuffling • 1. borders between exons and intronscoincide with the boundaries of different domains within proteins

33. 2. proteins composed of repeated unit are thought to arise through the exon duplication

34. 3. exons found in one gene turn out to be the recombination of exons in different otherwise genes

35. outline • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

36. RNA editing • Another way of altering the sequence of am mRNA • Two mechanism • 1. deamination • 2.guide RNA-directed uridine insertion or deletion

37. deamination • C U

38. As a consequence • Codon CAA could be changed into UAA (stop codon) • As a result • A full-length protein being truncated

39. Aslo A insoine • ADAR (adenosine deaminase acting on RNA) • Insoine could base pair with C • Thus, protein translated could altered

40. Uridine insertion • How? • Guide RNAs (gRNA) • Three domains • 1.5’ end, anchor, directs gRNA to the region of target RNA site for editing • 2.second, determines exactly where the Us will be inserted within the edited sequence • 3.3’ end ,poly-U stretch

41. Anchor region basie pair with target site • A residues are bulge out • Endonuclease cuts at mismatch • UTP added to be inserted in the blank region and linked by ligase

42. outline • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

43. mRNA transport • Three prerequisite for transportation out of the nucleus • 1. 5’-capped • 2.intron-free • 3.3’-polyadenylated

44. summary • 1.the chemistry of RNA splicing and the RNA splicing pathway, related spliceosome machinery • 2. diversities of self-splicing and difference and relationship among them • 3.the features and significances of alternative splicing , activation and repression of alternative splicing • 4.the evidences bolstering exon shuffling and its evolutionary means • 5.the mechanism and pathway of RNA editing