5. Pre-mRNA and ncRNA processing Flashcards
What is CTD tail in RNA pol II and why is it important?
C-terminal domain (CTD) tail of RNA pol II - coordinates recruitment of factors important for transcribed RNA processing - essential - cell wouldn’t survive without CTD
Which end of mRNA is capped? What is the cap’s function?
5’ end capped while mRNA still bound to Pol II:
- protects from degradation
- promotes pre-mRNA splicing
- needed for export out of nucleus
- needed for efficient translation - to recruit ribosome
RNAs from Pol I / Pol III - not capped
Explain mRNA capping process
After 20-40 nt synthesised 5’ cap is added while mRNA still bound to Pol II by capping enzyme:
1) 5’ triphosphate is cleaved
2) G residue added by 5’-5’ linkage (triphosphate linkage)
3) Cap G is methylated
If the capping not proper - mRNA degraded
Which end of mRNA is polyadenylated? Why is polyA tail important?
PolyA tail added to 3’ end while mRNA still bound to Pol II by enzymes recruited by CTD tail
PolyA tail needed for:
- stabilization
- protects from degradation
- allows export out of nucleus
Explain mRNA cleavage from RNA pol II and polyadenylation process
Cleavage and polyadenylation:
1) specific sequence at mRNA (polyA signal) end triggers recruitment of CPSF (cleavage and polyadenylation specificity factor) + CstF (cleavage stimulation factor)
2) once polyA signal transcribed - CPSF and CstF transferred to mRNA
3) additional cleavage enzymes - mRNA cleaved off from RNA pol II
4) PAP (poly-A polymerase) recruited - polyA added
5) PolyA tail length regulated -> mature mRNA 3’ end
What makes mRNA mature?
Splicing produces mature mRNA - introns excised out
Explain splicing reaction
Splicing reaction - two step catalytic process:
1) Intron :OH Nuc attack of splice site P -> break
2) 3’ :OH end attacks second splice site - exons joined - intron forms an intron lariat
What is an intron lariat?
In mRNA splicing - 2 :OH Nuc attacks excises out intron - intron forms a loop-ish structure - lariat
How are splice sites in mRNA recognized?
Sequence specific splice sites (5’ splice site + 3’ splice site) - proteins use guide RNAs base pair with targets - ribonucleoproteins (RNPs)
snRNPs (small): help recognize splice sites + cleave RNA + join exons
Explain the splicing process involving snRNPs
Splicing:
1) snRNP recognizes splice sites by base pairing
2) other snRNPs bind branch points and 3’ splice site
3) another snRNPs displaces -> makes one site bulge out
4) other snRNPs fold the intron bringing the exon ends closer -> catalyse cleavage joining exons
snRNPs: bridge and locate introns / exons for correct orientations for the catalysed reactions
How did splicing evolve?
Nuclear pre-mRNA splicing derived from self-cleaving introns - evolved to be more complicated -> alternative splicing - evolution of new proteins
How is it decided which exons are joined in alternative splicing?
Regulatory proteins - promote splicing at appropriate sites:
- SR proteins (serine and arginine rich) intronic/exonic splicing enhancers (ESE / ISE) - bind exons + splicing machinery
- intronic / exonic splicing silencers (ESS / ISS) heterogenous nRNPs (hnRNPs) bind exons but not splicing machinery
Is alternative splicing only for translated mRNA regions?
No, alternative splicing can affect both ORF and untranslated regions
What are the two mechanisms of alternative splicing?
Alternative splicing:
- general alternative splicing (AS): only exon splicing
- alternative last exon (ALE): exon + non-translated (UTR) splicing
How do mutations affect alternative splicing?
Mutations (even single nt change) - if normal splice site:
- mutated -> exon will be skipped in mRNA
- altered -> another splice site used (cryptic)
- mutated -> new splice site
What experimental methods must be completed to figure out if incorrect alternative splicing is causing disease?
Incorrect alternative splicing causing disease:
- take DNA sample -> **sequence **- identify exon splicing
- take RNA sample -> use PCR to confirm shorter/longer mRNA
- take protein sample in disease affected cells -> Western blot see if protein missing
What are microRNAs?
microRNAs (miRNAs) - class of ncRNAs:
- RNA Pol II transcripts
- processed in nucleus - exported into the cytoplasm
- functional unit - 22 nt mature sequence
- function: guides protein complex (RISC) to mRNAs - contributes to their translation and stability
What are miRNAs produced from?
miRNAs can be produced from exons / introns - coding / non-coding
How are miRNAs produced in splicing?
miRNAs can be produced from exons/introns - produced in mRNA splicing
Lecture summary
Quiz 1
Quiz 2
Quiz 3
What is the function of RISC protein?
RISC (RNA-induced silencing complex) - uses miRNA for recognizing complementary mRNA - activates RNase - cleaves the RNA
