Post transcriptional regulation

Question 1

Name pre-mRNA processing main steps.

Answer 1

1. 5’ capping
2. Cleavage at polyA site
3. Polyadenylation
4. RNA splicing

Question 1

How is the 5’ cap synthesized on mRNAs?

Answer 1

1. 5’ end of mRNA contains 5’ triphosphate
2. Gamma-phosphate is removed by phosphohydrolase
3. Guanylyl transferase transfers G from GTP to remaining phosphates at the 5’ end, forming 5’, 5’-triphosphate ester
4. Enzymes transfer methyl groups onto the 5’ end

Question 2

What are the two initial theories of polyadenylation?

Answer 2

1. Transcription stops at first poly(A) signal AAUAAA
2. Transcription stops at second poly(A) signal G/U

Research found that RNA is transcribed beyond the site of polyadenylation

Question 3

What are the steps to cleavage and polyadelyation of mRNA?

Answer 3

1. Cleavage and polyadenylation specificity factor (CPSF) binds to upstream PolyA signal
2. CStF interacts with downstream GU rich sequence, and with bound CPSF, forms loop in the RNA
3. Cleavage factor CF help stabilize the complex
4. PolyA polymerase PAP binds and stimulates cleavage at polyA site
5. Cleavage factor and product are released
6. PolyA binding protein PABPNI binding to inital short polyA tail accelerates PAP addition of residues to 3’ hydroxyl group generated by the cleavage reaction (polyadenylation)

Question 4

How does Pol. II terminate?

Answer 4

Pol II terminates close to polyA site.
Rat1 exonuclease is recruited by Rtt103 protein and degrades nascent RNA after cleavage and polyadenlylation, and induces a conformational change in Pol. II, destabilizing it.

Question 5

Explain the two transesterification reactions required for splicing.

Answer 5

1. Ester bond between 5’ of intron and 3’ of exon is exchanged for an ester bond with the 2’ of the branch site A residue.
2. Ester bond between 5’ phosphorus of exon 2 and 3’ of intron is exchanged for an ester bond with the 3’ of exon1

Essentially, we are exchanging phosphodiester bonds to splice out the intron.

Question 6

What are snRNPs formed of?

Answer 6

5 snRNAs (U1, 2, 3, 4, 5, 6) associate with 6-10 proteins forming small nuclear ribonucleoproteins

Question 7

Where does U1 and U2 snRNA bind with?

Answer 7

U1 binds to 5’ splicing site and U2 binds to branch point, but the A residue at the branch point does not pair.

Question 8

What is the role of snRNAs in splicing?

Answer 8

The two transesterification reactions of RNA splicing are catalyzed by spliceosome which is assembled using snRNPs assembled on the snRNAs

Question 9

What is U2AF?

Answer 9

U2AF is a U2-associated factor that binds to U2 snRNP and the 3’ end of an intron. With SF1, it promotes binding of U2 to branching point

Question 10

Explain the pre-mRNA splicing cycle.

Answer 10

1. U1 snRNA in U1 snRNP binds to 5’ splice site of pre-mRNA
2. U2AF and SF1 promote association of U2 snRNP with a branch point near the 3’ end of the intron
3. Binding of U1 and U2 recruits tri-snRNP complex (U4, 5, 6) during transition from the A to pre-B complex
4. U1 dissociates from B-complex
5. U4 disscociates as RNA helicase Brr2 pulls it away, causing the uniwinding of the U4/U6 duplex and dissociation of the associated proteins.
6. U6 changes conformation to interact with 5’ splice site and bind to two Mg+ ions, required for transesterification reactions.

Question 11

What is Brr2?

Answer 11

Brr2 is a helicase that promotes the release of U4 snRNA, causing unwinding of the U4/U6 duplex and the dissociation of the associated proteins

Question 12

What is the role of hmRNPs in splicing cycle?

Answer 12

Remain bound to spliced RNA to the exon-exon junction and is important for RNA export and nonsense-mediated decay after

Question 13

How does RNA processing prevents the accumulation of antisense transcripts? (RNAs where the transcription started in the wrong orientation?)

Answer 13

Higher frequency of polyA sites in antisense strand, such that polyA triggers transcription termination. PolyA sites downstream (correct orientation) is protected by U1 binding sites, which suppress cleavage and polyadenylation

Question 14

What are SR proteins?

Answer 14

Contain deomains rich in serine and arginine residues, crucial for protein-protein interactions.
Have RNA recognition motifs that allow them to bind to specific RNA sequences
Bind to exonic splicing enhancers, promotes inclusion of adjacent exons during alternative splicing
SR proteins, snRNAp and U2AF form cross-exon recognition complex, helps recognize exon-exon junctions during splicing process