8. Splicing Flashcards
What is the purpose of splicing?
How does it progress?
Removal of introns by endonucleolytic cleavage and ligation
Progresses largely in 5’-3’ direction but introns not necessarily spliced out in order - determined by RNA secondary structure and accessibility for spliceosome
How are introns defined?
Start with GT (donor) at 5’ of intron
End with AG (acceptor) at 3’
Where is the branch site? What does it contain?
Approximately 20-50 bp upstream of acceptor
Loosley conserved but always contains an adenine
What is the spliceosome formed of?
snRNAs bound to proteins = snRNPs
Major spliceosome formed of U1-4 + U6 snRNPs
Minor splicesome formed of U11, U12, U4atac, U5, U6atac snRNPs
Describe the 4 main steps in splicing
- Endonucleolytic cleavage of 5’ of intron (donor site, GT)
- Nucleophilic attack - GT forms lariat loop with A of branch site
- Endonucleolytic cleavage of 3’ of intron (acceptor, AG)
- Ligation of exons, introns degraded
How is splicing efficacy regulated?
- Consensus sites
- Trans-acting elements (proteins)
- Cis-acting elements (regulatory regions)
What is an ESS?
What’s its role?
Exonic splicing silencer
Silence adjacent splice sites and can cause exon skipping
Recruit proteins that negatively impact core splicing machinery by interfering with snRNAs so snRNPs aren’t formed
What is an ESE?
What’s its role?
Exonic splicing enhancer
Direct/enhances accurate splicing by recruiting serine/arginine (SR)-rich proteins which interact with U2 snRNPs to promote splicing
Give examples of how defects in splicing cause disease
- Disruption of splicing element:
a) Consenus donor/acceptor site
b) Branch site, e.g. COL5A1 and Ehlers Danlos type II
c) ESE/ESS, e.g. SMA (variant in ESE causes skipping of exon 7) and APC (removal of exon 14 –> truncated protein) - Toxic RNA - mutation increases stability of mRNA-protein complexes, e.g. DM1 - repetition of RBP binding sites, sequester RBP so less in cell for normal splicing
- Mutations affecting splicing factors, e.g TDP-43 in ALS
