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RNA splicing

RNA splicing. By xiao yi 生物学基地班 200431060010. In most cases of eukaryotic gene, the coding sequences is interrupted by noncoding sequences. The coding sequences are called exons The noncoding sequences are called introns.

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RNA splicing

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  1. RNA splicing By xiao yi 生物学基地班 200431060010

  2. In most cases of eukaryotic gene, the coding sequences is interrupted by noncoding sequences • The coding sequences are called exons • The noncoding sequences are called introns

  3. Before translation, the introns of pre-RNA must be removed, and this process is called RNA splicing

  4. RNA splicing: the process by which introns are removed from the pre-mRNA. • Alternative splicing : 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. 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.

  6. the exon-intron boundary at the 5’ end of the intron is called 5’ splicing site • the exon-intron boundary at the 3’ end of the intron is called 3’ splicing site

  7. Branch point site • an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

  8. The intron is removed in a Form Called a Lariat as the Flanking Exons are joined • RNA splicing is achieved by two successive transesterification reactions

  9. 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. 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. Exons from different RNA molecules can be fused by Trans-splicing • The only difference is that the other product---the lariat in the standard reaction---is, in trans splicing, is a Y shaped branch structure instead

  12. THE SPLICESOME MACHINERY • RNA splicing is carried out by a large complex called spliceosome • The spliceosome comprises about 150 proteins and 5 snRNAs • Many functions of the spliceosome are carried out by its RNA components.

  13. The five RNAs • U1, U2, U4, U5, and U6 are called small nuclear RNAs (snRNAs) • The complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNP)

  14. Three roles of snRNPs in splicing • They recognize the 5’ splicing site and the branch site • They bring those site together as required • They catalyze (or help catalyze) the RNA cleavage and joining reactions

  15. SPLICING PATHWAYS • Assembly • Rearrangement • catalysis

  16. 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

  17. 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. This A residue is available to react with the 5’ splice site.

  18. 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.

  19. 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 .This arrangement called the C complex.

  20. 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.

  21. 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 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.

  22. 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.

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

  24. When we examine the group 1 and group 2 self-splicing, we find the intron itself folds into a specific conformation within the precursor RNA and catalyze the chemistry of its own release

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

  26. Group I introns release a linear intron rather than a lariat • Instead of a branch point A residue, they use a free G nucleotide or nucleoside. This G species is bound by the RNA and its 3’ OH group is presented to the 5’ splicing site. Here fuses the G to the 5’ end of the intron. The freed 3’ end attacks the 3’ splicing site. In this case the intron is linear rather than a lariat structure

  27. Two kinds of splice-site recognition errors • Splice sites can be skipped. • “Pseudo” splice sites could be mistakenly recognized, particularly the 3’ splice site.

  28. Two ways to enhance the accuracy • RNA polymerase carries with it various proteins with roles in RNA processing • SR (serine argenine anthentic) proteins bind to sequences to called exonic splicing enhancers (ESEs) within the exons

  29. SR proteins are essential for splicing • They ensure the accuracy and efficiency of constitutive splicing. • They also regulate alternative splicing • They come in many varieties, some controlled by physical signals, others constitutively active. Some are expressed preferentially in certain cell types and control splicing in cell-type specific patterns.

  30. Alternative splicing • RNAs can be spliced in alternative ways to generate different mRNAs and, thus, different protein products

  31. There are five ways to splice a RNA

  32. 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

  33. Alternative splicing is regulated by activators and repressors • Proteins that regulate splicing bins to specific sites called exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS) • The former enhance and the latter repress, splicing at nearby splice sites

  34. The SR proteins bind RNA using one domain----RNA-recognition motif (RRM) • Each SR protein use RS domain which is rich in arginine and serine to mediate interactions between the SR protein and proteins within the splicing machinery

  35. Heterogeneous nuclear ribonucleoprotein (hnRNP) bind to RNA but lack the RS domain so cannot recruit splicing machinary, instead, they repress the use of those sites

  36. hnRNPI protein repress splicing by two ways: • hnRNPI bind to each end of exon, then interact with each other, looping out the exon • hnRNPI coat the RNA across the whole exon, making the exon invisible to the splicing machinary

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