Chapter 13 生物学基地班 孙钒 200431060017

1. Chapter 13生物学基地班 孙钒 200431060017 RNA Splicing

2. OUTLINE • The Chemistry of RNA Splicing • The Spliceosome Machinery • Splicing Pathways (important) • Alternative Splicing (important) • Exon Shuffling • RNA Editing • mRNA Transport

3. Key Words • Exon : coding sequences • Intron : intervening sequences • Pre-mRNA: the primary transcript of DNA • RNA splicing : process that intron are moved from the pre-RNA • Spliceosome : a huge molecular “machine” catalyse RNA splicing • Alternative splicing :some pre-mRNAs can be spliced in more than one way,and produce alternative mRNAs.

4. Topic 1 The Chemistry of RNA Splicing • Sequences within the RNA determine where splicing occurs • The intron is moved in a form called Lariat as the flanking exons are joined • Exons from different RNA molecules can be fused by trans-splicing

5. The borders between introns and exons are marked by specific nucleotide sequences ( GU—AG Law ) within the pre-mRNAs. Py-tract: rich in pyrimidine one: Sequences within the RNA determine where splicing occurs

6. Notice • GU in 5` splicing site, AG in 3` splicing site and A in branch point site are the most conserved sequences, and they are all in the intron. • These sequences are important for the distinguish between intron and exon ,remove of intron ,linkage of exons and delineate where splicing will occur.

7. Two :The intron is moved in a form called Lariat as the flanking exons are joined • RNA splicing consists of two successive transesterification reaction.

8. Reaction 1 : The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5’ splice site. As a result, the 5’ exon is released and the 5’-end of the intron forms a three-way junction structure.

9. Reaction 2 : The OH of the 5’ exon attacks the phosphoryl group at the 3’ splice site. As a consequence, the 5’ and 3’ exons are joined and the intron is liberated in the shape of a lariat.

10. The structure of three-way junction In addition to the 5` and 3` backbone linkages,a third phosphodiester extends from the 2`OH of that A to create a three-way junction.

11. Ensure the splicing only goes forward: One :an increase in entropy. Two :the excised intron lariat is rapidly degraded after its removal. Reaction 2

12. Notice • In the two reactions, there is no net gain in the number of chemical bonds. So no energy is demanded by the process. • But, we see a large amount of ATP is consumed during the splicing reaction. Why? This energy is required to properly assemble and operate the splicing machinery, not for the chemistry.

13. Three: Exons from different RNA molecules can be fused by trans-splicing • Trans-splicing: the process in which two exons carried on different RNA molecules can be spliced together. This process is rare .But all mRNAs in the nematode worm undergo trans-splicing.

14. Topic 2 : The Spliceosome Machinery • RNA splicing is carried out by a large complex called spliceosome • Spliceosome is a complex that mediates splicing of introns from pre-mRNA. And it comprises about 150 proteins and 5 snRNAs . Many functions of the spliceosome are carried out by its RNA components.

15. The five RNAs (U1, U2, U4, U5, and U6, 100-300 nt) are called small nuclear RNAs (snRNAs). • The complexes of snRNA and proteins are calledsmall nuclear ribonuclear proteins (snRNPs). • The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reaction. Different snRNPs come and go at different times,each carrying out particular functions in the reaction.

16. Three roles of snRNPs in splicing 1. Recognizing the 5’ splice site and the branch site. 2. Bringing those sites together. 3. Catalyzing (or helping to catalyze) the RNA cleavage. RNA-RNA, RNA-protein and protein-protein interactions are all important during splicing

17. RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA Branch-point binding protein

18. Topic 3 : Splicing Pathways • Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway • Self-splicing introns reveal that RNA can catalyze RNA splicing • Group I introns release a linear intron rather than a lariat • How does spliceosome find the splice sites reliably

19. One: Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway • Steps of splicing pathway: Assembly : step one 1. U1 recognize 5’ splice site. 2. One subunit of U2AF binds to Py tract and the other to the 3’ splice site. The former subunits interacts with BBP and helps it bind to the branch point. 3. Early (E) complex is formed

20. Assembly Steptwo 1.With the help of U2AF, U2 binds to the branch site replacing of BBP, and then A complex is formed. 2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded. This A residue is available to react with the 5’ splice site.

22. Assembly • Step three 1.U4, U5 and U6 form the tri-snRNPParticle. 2. With the entry of the tri-snRNP, the A complex is converted into the B complex.

23. Assembly • Step four 1 , U1 leaves the complex, and U6 replaces it at the 5’ splice site. 2 , U4 is released from the complex, allowing U6 to interact with U2 .This arrangement called the C complex.

24. Catalysis • Step one 1 , Formation of the C complex produces the active site, with U2 and U6 RNAs being brought together

25. 2 , Formation of the active site juxtaposes the 5’ splice site of the pre-mRNA and the branch site, allowing the branched A residue to attack the 5’ splice site to accomplish the first transesterfication reaction.

26. Catalysis • Step two U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3’-OH of the 5’ exon attacks the 3’ splice site. • Step three Release of the mRNA product and the snRNPs

28. Two : Self-splicing introns reveal that RNA can catalyze RNA splicing • Self-splicing introns: the intron itself folds into a specific conformation within the pre-mRNA and catalyzes the chemistry of its own release (recall RNA enzyme ) and the exon ligation. • Practical definition for self-splicing introns: the introns that can remove themselves from pre-RNAs in the test tube in the absence of any proteins or other RNAs. • Two classes of self-splicing introns, group I and group II self-splicing introns.

29. Three classes of RNA splicing

30. Notice :The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.

31. Three : Group I introns release a linear intron rather than a lariat • Instead of using a branch point A, group I introns use a free G to attack the 5’ splice site. • This G is attached to the 5’ end of the intron.The 3’-OH group of the 5’ exon attacks the 5’ splice site. • The two-step transesterification reactions are the same as that of splicing of the group II intron and pre-mRNA introns.

32. Three classes of RNA splicing

33. Group 1 intron structure Share a conserved secondary structure, which includes an “internal guide sequence” base-pairing with the 5’ splice site sequence in the upstream exon. • A complex secondary structure

34. Group 1 intron structure The tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor Bind any G-containing ribonucleotide.

35. Steps of group 1 intron splicing • free guanosine binds in the guanine-binding pocket • 3`OH of guanosine attacks phosphate at 5`end of intron • 3`OH of exon 1 attack 5`phosphate of exon 2 • Intron is released

36. Internal guide sequence base pairs with 5`end of intron • 3`guanosine binds in guanine-binding pocket • 3`OH of bound guanosine attacks phosphate to the right of IGS • Bond between 3`G and 5` phosphate hydrolyzes, leaving inactive intron

37. The similarity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterification

38. Four : How does spliceosome find the splice sites reliably • splice-site recognition is prone to 2 kinds of errors: ★Splice sites can be skipped. ★some site close in sequence but notlegitimate splice site ,could be mistakenly recognized. For example ,“Pseudo” splice sites could be mistakenly recognized and pair with component at 5`site, particularly the 3’ splice site.

39. Error produced by mistakes in splice-site selection

40. Two ways to enhance the accuracy of the splice-site selection 1 , the C-terminal tail of the RNA polymerase II carries various splicing proteins. when a 5`splice site is encountered in the newly synthesized RNA, those components are transferred from the Pol II C-terminal tail onto the RNA .

41. Once in place ,the 5`splice site components are poised to interact with those that bind to the next 3`splice siteto be synthesized. Thus ,the correct 3`splice site can be recognized before any competing sites further downstream have been transcribed. • This co-transcriptional loadingprocess greatly diminishes the likelihood of exon skipping.

42. 2 , a second mechanism guides against the use of incorrect sites by ensuring that splice sites close to exons are recognized preferentially. • SR proteins bind to sequences called exonic splicing enhancers (ESEs) within the exons. • SR bound to ESE interacts with components of the splicing machinery ,recruiting them to the nearby splicing sites. In this way , the machinery binds more efficiently to the nearby sites than to incorrect sites not close to exons.

43. SR protein recruits spliceosome components to the 5`and 3` splice sites SR proteins bind to ESEs ,recruit U2AF and U1snRNP to the downstream 5`and upstream 3`splice sites respectively. This initiates the assembly of the splice machinery on the correct sites and splice can proceed as outlined earlier.

44. SR proteins function • Ensure the accuracy and efficiency of constitutive splicing. • Regulate alternative splicing. They come in many varieties ,controlled by physiological signals and constitutively active. And some express preferential in certain cell types and control splicing in cell-type specific patterns.

45. Topic 4 : Alternative splicing • Single gene can produce multiple products by alternative splicing • Alternative splicing is regulated by activators and repressors • A small group of introns are spliced by an spliceosome composed of a different set of snRNPs

46. One : Single gene can produce multiple products by alternative splicing • Alternative splicing many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and ,different protein products.

47. As we know ,exons are not skipped and splice sites not ignored, why does alternative splicing occur so often ? Answer : some splice sites are used only some of the time ,leading to the production of different versions of the RNA from different transcripts of the same gene.

48. Five ways to splice an RNA

49. Alternative splicing can be either constitutive or regulated. Constitutive : more than one product from the same gene Regulated : different products are generated at different times, under different conditions , or in different cell or tissue type.

50. Constitutive alternative splicing high level SF2/ASF Splicing of the monkey SV40 T antigen RNA.