CSB349H1 Lecture Notes - Lecture 9: Alternative Splicing, Exon Skipping, Transesterification
14 May 2018
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Lecture 9(b): RNA Splicing
Spliceosome Mechanism:
• Pre-mRNA splicing catalyzed by assembly of snRNPs begins with:
o U1 snRNA binds to the 5’ splice site
o U2AF and BBP bind to the 3’ branch-point
[helper proteins]
§ U2 snRNA displaces BBP and U2AF to base-
pair with the branch-point site consensus sequences
• We have defined the 5’ splice site and
branch point at this stage
1. Creating the active site for the spliceosome:
o U4, U5 and U6 cooperate to create the active site
§ Active site positions the appropriate segments of
pre-mRNA for the first transesterification reaction
Ø Loops RNA and makes the 5’ splice site in close proximity
with the branch point
2. 1st transesterification reaction:
o U1 and U4 leave the complex
o 2’ OH group of branch site undergoes nucleophilic
attack with phosphoryl group of 5’ splice site
§ Lariat structure is formed
3. 2nd transesterification reaction:
o RNA-RNA arrangements form active site for the 2nd
transesterification to occur
o 3’ OH of 5’ exon undergoes nucleophilic attack with
phosphoryl group of 3’ splice site
4. Liberation & Merging:
o After the 2nd transesterification reaction the intron lariat
is released and spliceosome disassembles
§ Two exons will now merge together via formation
of phosphodiester bond
5. Debranching Enzyme:
o Debranching enzyme marks linear intron from lariat and be immediately degraded
Alternative Splicing:
• Exons found within a primary RNA construct often encode a specific domain in a protein
o Alternative splicing allows for the ‘skipping’ of certain exons (i.e. certain domains of
a protein) to generate a different protein with altered function and structures
ð Variety of different alternative splicing
Document Summary
Lecture 9(b): rna splicing: pre-mrna splicing catalyzed by assembly of snrnps begins with, u1 snrna binds to the 5" splice site, u2af and bbp bind to the 3" branch-point. Active site positions the appropriate segments of pre-mrna for the first transesterification reaction. Two exons will now merge together via formation of phosphodiester bond: debranching enzyme, debranching enzyme marks linear intron from lariat and be immediately degraded. Alternatively spliced mrna products are always seen in all cell types due to the ambiguity of splice sites (equally recognized and randomly utilized: regulative alternative splicing, alternative mrnas are generated depending on the context. Alternatively spliced mrna products are not always seen; depends on cell type or different developmental stage exclusive. Alternative splicing patterns are not well conserved between species: can be considered a mechanism for speciation. Function to interact with spliceosome and help recruit machinery to introns: intronic splicing enhancers (ises)
