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CHAPTER 13

CHAPTER 13. RNA SPLICING. 生物学基地班 200431060026 黎浩. OUTLINE. THE CHEMISTRY OF RNA SPLICING THE SPLICEOSOME MACHINERY SPLICING PATHWAYS ALTERNATIVE SPLICING EXON SHUFFLING RNA EDITING mRNA TRANSPOTR. THE CHEMISTRY. Sequences within the RNA Determine Where Splicing Occurs.

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CHAPTER 13

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  1. CHAPTER 13 RNA SPLICING 生物学基地班 200431060026 黎浩

  2. OUTLINE • THE CHEMISTRY OF RNA SPLICING • THE SPLICEOSOME MACHINERY • SPLICING PATHWAYS • ALTERNATIVE SPLICING • EXON SHUFFLING • RNA EDITING • mRNA TRANSPOTR

  3. THE CHEMISTRY Sequences within the RNA Determine Where Splicing Occurs • How are the introns and exons distinguished from each other? • How are introns removed? • How are exons joined with high precision?

  4. The consensus sequences for human The borders between introns and exons are marked by specific nucleotide sequencese within the pre-mRNAs.These sequences delineate where splicing will ocuurs.

  5. 5’splice site : the exon-intron boundary at the 5’ end of the intron • 3’ splice site : the exon-intron boundary at the 3’ end of the intron • Branch point site : an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract

  6. The intron is removed in a Form Called a Lariat as the Flanking Exons are joined • Splicing is achieved by two successive transesterification reactions.Phosphodiester linkages within the pre-mRNA are broken and new ones are formed.

  7. Step 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. • Notice that the 5’ exon is aleaving group in the first transesterification.

  8. Step 1

  9. The structure of the three-way junction formed during the splicing reaction

  10. Step 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.

  11. Step 2

  12. Another point about the splicing reaction is direction: splicing only goes forward. Two features are as follows. First, the forward reaction involves an increase in entropy-a single pre-mRNA molecule is split into two molecules, the mRNA and the liberated lariat. Second, the excised intron is rapidly degraded after its removal and so is not available to partake in the reverse reaction.

  13. 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. • The chemistry of this standard splicing reaction described previously, and the spliced product is indistinguishable.

  14. THE SPLICING MACHINERY RNA Splicing Is Called Out by a Large Complex Called the Splicing • The transesterification reactions just described are mediated by a huge molecule “machine” called the splicesome. This complex comprises about 150 proteins and is similar in size to a ribosome.

  15. The five RNAs (U1,U2,U4,U5,and U6) are collectively called small nuclear RNAs (snRNAs). Each of these RNAs is between 100 and 300 nucleotides long and is complexed with several proteins. These RNA-protein complexes are called small nuclear ribonuclear proteins (snRNPs-pronounced “snurps”).

  16. Three roles in splicing of the snRNAs: • Recognizing the 5’ splice site and the branch site. • Bringing those sites together. • 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 Different snRNAs recognize the same sequence in the pre-mRNA at different stages of the splicing reaction, as shown here for U1 and U6 recognizing the 5’ splice site.

  18. RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA SnRNP U2 is shown recognize the branch site.

  19. RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA The RNA:RNA pairing between the snRNPs U2 and U6 is shown.

  20. RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA The same sequence within the pre-mRNA is recognized by a protein at one stage and displayed by an snRNP at another.

  21. SPLICING PATHWAYS Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway • Initially, the 5’ splice site is recognized by the U1 snRNP. • 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. • This arrangement of proteins and RNA is called the Early complex.

  22. Then, snRNP binds to the branch site, aided by U2AF and display BBP. This arrangement is called the A complex. • 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.

  23. The next step is a rearrangement of the A complex to bring together all three splice sites. This is achieved as follows: the U4 and U6 snRNPs, along with the U5snRNP, join the complex. With the entry of the tri-snRNP, the A complex is converted to B complex.

  24. In the next step, U1 leaves the complex, and U6 replaces it at the 5’ splice site. • U4 is released from the complex, allowing U6 to interact with U2 .This arrangement called the C complex.

  25. Step of the spliceosome-mediated splicing reaction

  26. Self-splicing introns reveal that RNA can catalyze RNA splicing • Self-splicing: The intron itself folds into a specific conformation within the precursor RNA and catalyzes the chamistry of its own release. • In the terms of a practical definition, self-splicing means that these introns can remove themselves from RNAs in the test tube in the absence of any proteins or other RNA moleculars.

  27. Three class of RNA Splicing

  28. 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.

  29. 1. Smaller than group II introns 2. Share a conserved secondary structure, which includes an “internal guide sequence” base-pairing with the 5’ splice site sequence in the upstream exon. 3. The tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor

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

  31. How does spliceosome find the splice sites reliably • Splice-site recognition is prone to two kinds of errors. • First, splice sites can be skipped. • Second, other sites, close in sequence but not legitimate splice sites.

  32. Errors produced by mistakes in splice-site selection

  33. Two ways in which the accuracy of splice-site selection can be enhanced are as follows. • First, while transcribing a gene to produce the RNA, RNA polymerase II carries with it various proteins with roles in RNA processing. • Second, the splice sites close to exons are recognized preferentially. SR proteins bind to the ESEs (exonic splicing enhancers) present in the exons and promote the use of the nearby splice sites by recruiting the splicing machinery to those sites

  34. SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites.

  35. SR proteins are essential for splicing • Ensure the accuracy and efficacy of constitutive splicing • Regulate alternative splicing • There are many varieties of SR proteins. Some are expressed preferentially in certain cell types and control splicing in cell-type specific patterns

  36. 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. Single genes can produce multiple products by alternative splicing

  37. Five different ways to alternatively splice a pre-mRNA

  38. Five ways to splice an RNA : By including all exons, an mRNA containing all three exons is generated. Exon skipping gives an mRNA containing just exons 1 and 3. By exon extension, part of intron 1 is included together with the three exons. In another case, a complete intron is retained in the mature mRNA. Finally, exons 2 and 3 might be used as alternatives, generating a mixture of mRNAs, each including exon 1 and either exon 2 or 3.

  39. An example of constitutive alternative splicing :Splicing of the SV40 T antigen RNA

  40. Alternative Splicing Is Regulated by Activators and Repressors • Proteins that regulate splicing bind to specific sites called exonic (or intronic) splicing enhances (ESE or ISE) or silencers (ESS and ISS). • The former enhance, and the latter repress, splicing at nearly splice sites. • SR protein can determine whether a particular splice site is used in a particular cell type or at a particular stage of development.

  41. hnRNPs binds RNA and act as repressor Most silencers are recognized by hnRNP ( heterogeneous nuclear ribonucleoprotein) family. These proteins bind RNA, but lack the RS domains. Therefore, (1) They cannot recruit the splicing machinery. (2) they block the use of the specific splice sites that they bind.

  42. Some alternatively spliced exons appear in mRNA unless prevented from doing so by a repressor protein (shown in part a). Others appear only if a specific activator promotes their inclusion (part b). Either mechanism can be used to regulate splicing such that in one cell type a particular exon is included in an mRNA, whereas in another it is not.

  43. hnRNPs binds RNA and act as repressors • Most silencers are recognized by hnRNP ( heterogeneous nuclear ribonucleoprotein) family. • These proteins bind RNA, but lack the RS domains. Therefore, (1) They cannot recruit the splicing machinery. (2) they block the use of the specific splice sites that they bind.

  44. Inhibition of splicing by hnRNAI

  45. A small group of intron are spliced by minor spliceosome • Higher eukaryotes use the major splicing machinery we have discussed thus far to direct splicing of the majority of their pre-mRNA. • U11 and U12 components of the alternative splicesome have the same roles in the splicing reaction as U1 and U2 of the major form, but they recognize distinct sequences.

  46. The AT-AC splicesome catalyzed splicing

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