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BIS477 World of RNA > Co-transcriptional RNA processing > Flashcards

Co-transcriptional RNA processing Flashcards

1
Q

What is the Pol II CTD? (3)

A
  • 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
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2
Q

Which RNAs are made by RNA polymerase II? (2)

A
  • mRNA
  • lncRNA (long non-coding)
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3
Q

What are the main steps of gene expression? (5)

A
  • Transcription
  • 5’ capping
  • Splicing to remove introns
  • 3’ polyadenylation
  • Export to the cytoplasm for translation
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4
Q

When does 5’ capping occur? (2)

A
  • Very early on during transcription when mRNA is approximately 20 bases long
  • Capping enzymes bind to RNA polymerase II which couples capping to transcription
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5
Q

What enzymes are involved in 5’ capping? (4)

A
  • RNA triphosphatase (RTPase)
  • RNA guanylyltransferase (GTase)
  • RNA (guanine-N7)-methyltransferase (N7MTase)
  • RNA (nucleoside-2’-O-)-methyltransferase (2’OMTase)
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6
Q

What is the 5’ cap? (3)

A
  • 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
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7
Q

What happens if any of the processing steps go wrong?

A

mRNA will be degraded before it can be exported out of the nucleus

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8
Q

What are the key steps in splicing? (6)

A
  • 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
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9
Q

What are snRNPs? (2)

A
  • Small nuclear ribonucleoproteins
  • Form the spliceosome
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10
Q

What happens during formation of the 3’ end of mRNA? (4)

A
  • 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
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11
Q

What proteins are involved in 3’ end processing? (6)

A
  • 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
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12
Q

Why do you need to define exons?

A

Introns are much bigger than exons so need to make them easy to find for splicing

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13
Q

Which proteins help to define exon boundaries? (2)

A
  • hnRNP proteins
  • SR proteins
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14
Q

What highly conserved sequences define splice sites? (2)

A
  • 5’ splice site GU
  • 3’ splice site AG
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15
Q

How does processing occur co-transcriptionally? (2)

A
  • 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
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16
Q

What is the pol II CTD sequence?

A

Tyrosine, serine, proline, threonine, serine, proline, serine (YSPTSPS)

17
Q

What is the RNA poly II CTD required for? (3)

A
  • Essential for splicing
  • Essential for 3’ end processing
  • Phosphorylation of the CTD governs interaction with these processing
18
Q

What is triggered by Ser5 phosphorylation of the CTD?

A

Spliceosome association

19
Q

What happens if you inhibit CTD phosphorylation with DRB? (2)

A
  • Inhibits co-transcriptional processing but not processing uncoupled from transcription
  • DRB inhibits Cdk9
20
Q

What are the serine CTD modifications? (3)

A
  • 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
21
Q

How is the CTD modified?

A

Modification is dynamic during transcription

22
Q

How is the pol II CTD phosphorylation linked to 5’ capping? (3)

A
  • Poll II CTD gets phosphorylated on ser5 by Cdk7
  • Triggers recruitment of capping enzyme to the CTD
  • Leads to cap methylation
23
Q

How is CTD phosphorylation linked to splicing? (2)

A
  • 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
24
Q

How is CTD phosphorylation linked to 3’ end formation? (3)

A
  • 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
25
Q

How is CTD phosphorylation linked to transcriptional checkpoints? (3)

A
  • Checkpoints slow down the poly II to allow time for proper spliceosome assembly
  • Checkpoints are associated with Ser5 phosphorylation which causes pausing of poly II and stimulates spliceosome recruitment
  • If the checkpoint is satisfied, transcription continues and more pSer2 accumulates during elongation
26
Q

How does RNA processing influence transcription termination? (3)

A
  • Transcription is suppressed beyond the normal polyA site
  • Mutation of the polyA site allows transcription to continue
  • Xrn2 exonuclease binds to free non-capped 5’ end of mRNA generated after 3’ end cleavage and degrades it and eventually removes pol II from the gene to stop transcription past the polyA site (torpedo model for transcription termination)

BIS477 World of RNA (1 decks)

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