Coupling of mRNA processing to transcription

Question 1

What is the Pol II CTD? (3)

Answer 1

• 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

Question 2

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

Answer 2

• mRNA
• lncRNA (long non-coding)

Question 3

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

Answer 3

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

Question 4

When does 5’ capping occur? (2)

Answer 4

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

Question 5

What is the 5’ cap? (3)

Answer 5

• 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

Question 6

What happens if any of the processing steps go wrong?

Answer 6

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

Question 7

What are the key steps in splicing? (6)

Answer 7

• 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

Question 8

What are snRNPs? (2)

Answer 8

• Small nuclear ribonucleoproteins
• Form the spliceosome

Question 9

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

Answer 9

• 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

Question 10

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

Answer 10

• 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

Question 11

Why do you need to define exons?

Answer 11

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

Question 12

Which proteins help to define exon boundaries? (2)

Answer 12

• hnRNP proteins
• SR proteins

Question 13

What highly conserved sequences define splice sites? (2)

Answer 13

• 5’ splice site GU
• 3’ splice site AG

Question 14

How does processing occur co-transcriptionally? (2)

Answer 14

• 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

Question 15

What is the pol II CTD sequence?

Answer 15

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

Question 16

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

Answer 16

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

Question 17

What is triggered by Ser5 phosphorylation of the CTD?

Answer 17

Spliceosome association

Question 18

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

Answer 18

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

Question 19

What are the serine CTD modifications? (3)

Answer 19

• 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

Question 20

How is the CTD modified?

Answer 20

Modification is dynamic during transcription

Question 21

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

Answer 21

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

Question 22

How is CTD phosphorylation linked to splicing? (2)

Answer 22

• 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

Question 23

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

Answer 23

• 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

Question 24

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

Answer 24

• 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

Question 25

How is transcription terminated? (2)

Answer 25

• 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)

Question 26

What is phosphorylated serine 2 on the CTD a marker of? (2)

Answer 26

• Elongation of the RNA during transcription
• pSer2 builds up as the RNA as transcribed after passing early checkpoints