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Chapter 13 RNA splicing

Chapter 13 RNA splicing. The chemistry of RNA splicing The spliceosome machinery Splicing pathways Alternative splicing Exon shuffling RNA editing mRNA transport. The chemistry of RNA splicing. Sequences within the RNA determine where splicing occurs.

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Chapter 13 RNA splicing

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  1. Chapter 13 RNA splicing • The chemistry of RNA splicing • The spliceosome machinery • Splicing pathways • Alternative splicing • Exon shuffling • RNA editing • mRNA transport

  2. The chemistry of RNA splicing

  3. Sequences within the RNA determine where splicing occurs

  4. The intron is removed in a form called a lariat as the flanking exons are joined

  5. Fig 13-5 The structure of the three-way junction formed during the splicing reaction

  6. The splicing reactions have no net gain in the no. of chemical bonds. Yet, a large number of ATP is consumed, not for the chemistry, but to properly assemble and operate the slicing machinery. • What ensures the slicing only goes forward? • Increase in entropy • Excised intron is quickly degraded

  7. Box 13-1 Adenovirus and the discovery of splicing Map of the human adenovirus-2 genome Human adenovirus is a DNA virus which serve as a model for studying eukaryotic gene regulation. Same promoter and the tripartate leader sequence for all the transcripts which are resulted from alternative splicing

  8. R-loop mapping of the adenovirus-2 late messenger RNAs.

  9. Exons from different RNA molecules can be fused by trans-splicing Fig 13-6 trans-splicing Although generally rare, trans-spicing occurs in almost all the mRNAs of trypanosomes (錐體蟲) eg. Nematode worms (C. elegans)

  10. The spliceosome machinery • RNA splicing is carried out by a large complex called the spliceosome • Spliceosome comprises about 150 proteins and 5 RNAs and is similar in size with ribosomes. • Many functions of spliceosome are carried out by RNAs rather by proteins. • Five RNAs (U1, U2, U4, U5 and U6) are collectively called small nuclear RNAs (snRNAs)with the size in the range of 100-300 bp long and are complexed to • small nuclear ribonuclear proteins (snRNP) • The makeup of spliceosome varies at different stages of the splicing reaction.

  11. Fig 13-7 some RNA-RNA hybrids formed during the splicing reaction

  12. Structure of spliceosomal protein-RNA complex: U1A binds hairpin II of U1 snRNA

  13. Splicing pathways • Assembly, rearrangements, and catalysis within the spliceosome: the splicing pathway

  14. Early (E) complex Fig 13-8 steps of the spliceosome-mediated splicing reaction. The A complex The B complex U1 snRNP replaced by U6 snRNP Active site formed U4 released, U2 forming RNA-RNA hybrids with U6 Lariat initially still bound with tri-snRNP. Soon the lariat degrades, leaving the snRNPs to be recycled.

  15. Self-splicing introns reveal that RNA can catalyze RNA splicing

  16. Fig 13-9 group I and group II introns Group I introns release a linear intron rather than a lariat

  17. Fig 13-10 Proposed folding of the RNA catalytic regions for splicing of group II introns and pre-mRNAs.

  18. Group I intron use a free G nucleoside or nucleotide. This G species is bound by the RNA and its 3’OH group is presented to the 5’ splice site. • Structure of group I intron includes • a binding pocket that will accommodate any guanine nucleoside and nucleotide. • Internal guide sequence that base-pairs with the 5’ splice site sequence.

  19. Box 13-1 group I introns can be converted into true ribozymes.

  20. How does the spliceosome find the splice sites reliably? • The average exon in only some 150 nt long, whereas the average intron is about 3,000 nt long. Thus, the exons must be identified within a vast ocean of intronic sequences.

  21. Fig 13-12 Errors produced by mistakes in splice-site selection.

  22. To avoid exon-skipping: co-transcriptional loading of spliceosome components onto the splice sites.(refer to 12-20) • To avoid pseudo splice-site selection:

  23. Fig 13-12 SR (serine-arginine rich) proteins recruit spliceosome components to the 5’ and 3’ splice sites. ESE: exonic splicing enhancer

  24. A small group of introns are spliced by an alternative spliceosome composed of a different set of snRNPs Fig 13-13 The AT-AC (minor) spliceosome catalyzed splicing. This minor spliceosome has distinct splice site sequences but same splicing chemistry.

  25. Alternative Splicing • Single genes can produce multiple products by alternative splicing • By alternative splicing multiple proteins can be produced from a single gene. These different proteins are called isoforms. They can have similar functions, distinct functions, or even antagonistic functions.

  26. Fig 13-14 Alternative splicing in the troponin T gene

  27. Fig 13-15 Five ways to splice a RNA

  28. Fig 13-16 constitutive alternative splicing: monkey virus SV40 T-antigen 5’SST: 5’ splice site used to generate the large T mRNA 5’sst: 5’ splice site used to generate the small T mRNA

  29. Several mechanisms exist to ensure mutually exclusive splicing • Steric hindrance • Combinations of major and minor splice sites 3. Nonsense-mediated decay

  30. The curious case of the Drosophila Dscam gene: Mutually exclusive splicing on a grand scale

  31. Dscam Down syndrome cell-adhesion molecule

  32. Alternative splicing is regulated by activators and repressors Fig 13-22 Regulated alternative splicing • Proteins that regulate splicing bind to specific sites called exonic (intronic) splicing enhancers (ESE/ISE) or silencers (ESS/ISS)

  33. Fig 13-23 mammalian splicing repressor hnRNPI

  34. Fig 13-25 a cascade of alternative splicing events determines the sex of a fly. Sxl: splicing repressor Tra: splicing activator

  35. Exon Shuffling • Exons are shuffled by recombination to produce genes encoding new proteins • Why introns are present in all organisms except bacteria??? Introns early model: due to selection pressure to speed chromosome replication and cell division Introns late model: due to a transposome like mechanism Why have the introns been retained in (higher) eukaryotes?? The advantages of exon shuffling

  36. Fig 13-26 Exons encode protein domains.

  37. Fig 13-27 Genes made up of parts of other genes.

  38. Fig 13-28 Accumulation, loss, and reshuffling of domains during the evolution of the family of chromatin modifying enzymes.

  39. Evidences of exon shuffling • The boundaries between exons/introns often coincide with boundaries between domains. • Some proteins (eg. immunoglobulin) have repeating units, which might be due to gene duplications. • Related exons are sometimes found in unrelated genes.

  40. RNA Editing • Site-specific deamination • Guide RNA-directed uridine insertion or deletion (often in trypanosomes and mitochondria).

  41. Human apolipoprotein gene Fig 13-29 RNA editing by deamination:

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