Coupling of mRNA processing to transcription Flashcards
What is the Pol II CTD? (3)
- Carboxy terminal domain part of the Rpb1 subunit (largest subunit of RNA polymerase II)
- In humans, made of 52 repeats of the aa sequence YSPTSPS
- Budding yeast have 26 repeats, correlation between repeat number and organism complexity
Which RNAs are made by RNA polymerase II? (2)
- mRNA
- lncRNA (long non-coding)
What are the main steps of gene expression? (5)
- Transcription
- 5’ capping
- Splicing to remove introns
- 3’ polyadenylation
- Export to the cytoplasm for translation
When does 5’ capping occur? (2)
- Very early on during transcription when mRNA is approximately 20 bases long
- Capping enzymes bind to RNA polymerase II which couples capping to transcription
What is the 5’ cap? (3)
- 7-methylguanosine (m7g) cap linked to the first nucleotide via a triphosphate linkage
- Capped mRNA is stable
- Enhances mRNA export and translation in the cytoplasm
What happens if any of the processing steps go wrong?
mRNA will be degraded before it can be exported out of the nucleus
What are the key steps in splicing? (6)
- U1 snRNP binds to 5’ splice site
- U2AF and branch-point binding protein (BBP) bind to branch-point site
- U2 snRNP displaces BBP and U2AF at the branch-point site
- U4/U5/U6 triple snRNP joins
- U1 and U4 leave which leaves a catalytically active lariat structure
- 5’ end of the intron is cleaved, then 3’ end which releases the lariat structure and the exons are joined together
What are snRNPs? (2)
- Small nuclear ribonucleoproteins
- Form the spliceosome
What happens during formation of the 3’ end of mRNA? (4)
- mRNA is cleaved at the CA sequence which is 10-30 nucleotides downstream from the polyadenylation signal (AAUAAA)
- CA sequence is upstream of GU/U-rich sequence element which aids in cleavage but ultimately gets degraded in the nucleus
- ~250 A’s added to the end of the mRNA after cleavage to make polyA tail
- Important for mRNA export and translation
What proteins are involved in 3’ end processing? (6)
- Proteins bind to RNA polymerase II which couples processing with translation
- CPSF recognises the AAUAAA polyadenylation signal
- CFI and CFII cleave the mRNA at CA sequence
- Rearrangement of the complex, CPSF stays on
- PAP adds the polyA tail
- PABP binds to the polyA tail
Why do you need to define exons?
Introns are much bigger than exons so need to make them easy to find for splicing
Which proteins help to define exon boundaries? (2)
- hnRNP proteins
- SR proteins
What highly conserved sequences define splice sites? (2)
- 5’ splice site GU
- 3’ splice site AG
How does processing occur co-transcriptionally? (2)
- RNA polymerase II CTD acts as a scaffolding platform for splicing factors, capping enzymes and cleavage polyA factors to bind to so they are ready to perform their actions while the RNA is being transcribed
- U1 snRNP binds directly to RNA polymerase II
What is the pol II CTD sequence?
Tyrosine, serine, proline, threonine, serine, proline, serine (YSPTSPS)
What is the RNA poly II CTD required for? (3)
- Essential for splicing
- Essential for 3’ end processing
- Phosphorylation of the CTD governs interaction with these processing
What is triggered by Ser5 phosphorylation of the CTD?
Spliceosome association
What happens if you inhibit CTD phosphorylation with DRB? (2)
- Inhibits co-transcriptional processing but not processing uncoupled from transcription
- DRB inhibits Cdk9
What are the serine CTD modifications? (3)
- Serine 2 is phosphorylated by Cdk9, Cdk12 and Cdk13 during elongation
- Serine 5 is phosphorylated by Cdk7 for RNA capping and splicing
- Serine 7 is widely phosphorylated by Cdk7 but function unclear
How is the CTD modified?
Modification is dynamic during transcription
How is the pol II CTD phosphorylation linked to 5’ capping? (3)
- Poll II CTD gets phosphorylated on ser5 by Cdk7
- Triggers recruitment of capping enzyme to the CTD
- Leads to cap methylation
How is CTD phosphorylation linked to splicing? (2)
- Triggers recruitment of U2AF65 and 35 (U2AF dimer) which recognise the branch point sequence (near 3’ splice site)
- Triggers recruitment of PRP19C (ubiquitin ligase) which is responsible for eviction of U4 snRNP from the spliceosome leaving it catalytically active
How is CTD phosphorylation linked to 3’ end formation? (3)
- CPSF accompanies pol II
- Transcription of the polyA site causes pol II to pause which triggers phosphorylation of CTD ser2 by Cdk12
- This triggers recruitment of CSTF which stimulates mRNA cleavage
How is CTD phosphorylation linked to transcriptional checkpoints? (3)
- Checkpoints slow down the poly II to allow time for proper spliceosome assembly
- Early checkpoint associated with Ser5 phosphorylation which causes pausing of poly II and stimulates spliceosome recruitment
- If the checkpoint is satisfied (proper pSer5 and spliceosome assembly), transcription continues and more pSer2 accumulates during elongation
How is transcription terminated? (2)
- Xrn2 exonuclease binds to free non-capped 5’ end of mRNA generated after 3’ end cleavage and degrades it
- Pol II remains bound and transcribing so Xrn2 eventually displaces pol II from the gene to stop transcription (torpedo model for transcription termination)
What is phosphorylated serine 2 on the CTD a marker of? (2)
- Elongation of the RNA during transcription
- pSer2 builds up as the RNA as transcribed after passing early checkpoints
