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Ch 12 : Mechanisms of Transcription Ch 13 : RNA Splicing Ch 14 : Translation

Ch 12 : Mechanisms of Transcription Ch 13 : RNA Splicing Ch 14 : Translation Ch 15 : The Genetic code. Molecular Biology Course. CHAPTER 13 RNA Splicing. Primary transcript. Figure 13-1.

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Ch 12 : Mechanisms of Transcription Ch 13 : RNA Splicing Ch 14 : Translation

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  1. Ch 12 : Mechanisms of Transcription Ch 13 : RNA Splicing Ch 14 : Translation Ch 15 : The Genetic code

  2. Molecular Biology Course CHAPTER 13 RNA Splicing

  3. Primary transcript Figure 13-1

  4. Most of the eukaryotic genes are mosaic (嵌合体), consisting of intervening sequences separating the coding sequence • Exons (外显子): the coding sequences • Introns (内含子) : the intervening sequences • RNA splicing: the process by which introns are removed from the pre-mRNA. • Alternative splicing (可变剪接): some pre-mRNAs can be spliced in more than one way , generating alternative mRNAs. 60% of the human genes are spliced in this manner.

  5. CHAPTER 13 RNA Splicing Topic 1 : THE CHEMISTRY OF RNA SPLICING

  6. The chemistry of RNA splicing Sequences within the RNA Determine Where Splicing Occurs The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.

  7. The consensus sequences for human Figure 13-2

  8. 5’splice site (5’剪接位点): the exon-intron boundary at the 5’ end of the intron • 3’ splice site (3’剪接位点): the exon-intron boundary at the 3’ end of the intron • Branch point site (分枝位点): an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

  9. The chemistry of RNA splicing The intron is removed in a Form Called a Lariat (套马索) as the Flanking Exons are joined Two successivetransesterification: 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.

  10. Figure 13-3 Three-way junction

  11. The structure of three-way function Figure 13-4 This figure has an error

  12. 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.

  13. Figure 13-3

  14. The chemistry of RNA splicing 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.

  15. Trans-splicing Figure 13-5 Not a lariat

  16. CHAPTER 13 RNA Splicing Topic 2 THE SPLICESOME MACHINERY

  17. The spliceosome machinery RNA splicing is carried out by a large complex called spliceosome • The above described splicing of introns from pre-mRNA are mediated by thespliceosome. • The spliceosome comprises about 150 proteins and 5 snRNAs. • Many functions of the spliceosome are carried out by its RNA components.

  18. 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 (snRNP, pronounces “snurps”). • The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reaction

  19. 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.

  20. RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA Figure 13-6

  21. CHAPTER 13 RNA Splicing Topic 3 SPLICING PATHWAYS

  22. Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway (Fig. 13-8) • Assembly step 1 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 Splicing pathways

  23. Assembly step 2 1. U2 binds to the branch site, 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(Figure 13-6). This A residue is available to react with the 5’ splice site.

  24. E complex Figure 13-8 A complex Figure 13-6b

  25. Assembly step 3 1.U4, U5 and U6 form the tri-snRNP Particle. 2. With the entry of the tri-snRNP, the A complex is converted into the B complex.

  26. A complex B complex Figure 13-8

  27. Assembly step 4 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 (Figure 13-6c).This arrangement called the C complex.

  28. B complex C complex in which the catalysis has not occurred yet Figure 13-6c Figure 13-8

  29. Catalysis Step 1: Formation of the C complex produces the active site, with U2 and U6 RNAs being brought together Formation of the active site juxtaposes (并置) 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 (转酯) reaction.

  30. Catalysis Step 2: 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. • Final Step: Release of the mRNA product and the snRNPs

  31. C complex 1st reaction Figure 13-8 2nd reaction

  32. splicesome-mediated splicing reactions E complex A complex B complex Figure 13-8 C complex （没有该complex的图）

  33. How does spliceosome find the splice sites reliably Splicing pathways Two kinds of splice-site recognition errors • Splice sites can be skipped. • “Pseudo” splice sites could be mistakenly recognized, particularly the 3’ splice site.

  34. Figure 13-12

  35. Reasons for the recognition errors (1) The average exon is 150 nt, and the average intron is about 3,000 nt long (some introns are near 800,000 nt) • It is quite challenging for the spliceosome to identify the exons within a vast ocean of the intronic sequences.

  36. (2) The splice site consensus sequence are rather loose. For example, only AGG tri-nucleotides is required for the 3’ splice site, and this consensus sequence occurs every 64 nt theoretically.

  37. Two ways to enhance the accuracy of the splice-site selection 1. Because the C-terminal tail of the RNA polymerase II carries various splicing proteins, co-transcriptional loading of these proteins to the newly synthesized RNA ensures all the splice sites emerging from RNAP II are readily recognized, thus preventing exon skipping.

  38. 2. There is a mechanism to ensure that 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

  39. Figure 13-13 SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites.

  40. 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

  41. CHAPTER 13 RNA Splicing Topic 4ALTERNATIVE SPLICING

  42. Single genes can produce multiple products by alternative splicing • Many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products. Alternative splicing

  43. Drosophila DSCAM gene can be spliced in 38,000 alternative ways Figure 13-13

  44. There are five different ways to alternatively splice a pre-mRNA Figure 13-15

  45. Alternative splicing can be either constitutive or regulated • Constitutive alternative splicing: more than one product is always made from a pre-mRNA • Regulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types

  46. An example of constitutive alternative splicing :Splicing of the SV40 T antigen RNA Figure 13-16

  47. Alternative splicing is regulated by activators and repressors • The regulating sequences :exonic (or intronic) splicing enhancers (ESE or ISE) orsilencers (ESS and ISS). The former enhance and the latter repress splicing. • Proteins that regulate splicing bind to these specific sites for their action Alternative splicing

  48. SR proteins binding to enhancers act as activators. (1) One domain is the RNA-recognition motif (RRM) (2) The other domain is RS domain rich in arginine and serine. This domain mediates interactions between the SR proteins and proteins within the splicing machinery.

  49. 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.

  50. Regulated alternative splicing Figure 13-17

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