Lecture 10: RNA Structure & Processing (Post-Transcription) Part 2

Question 1

What does RRM stand for and what is its function

Answer 1

RRM = RNA recognition motif
- it is a structure that provides a way to recognize/interact with RNA aside from the ends

Question 2

What is the RRM Domain Structure?

Answer 2

• Two α helices (green) and four β strands (red)
• Conserved RNP1 and RNP2 regions – located in the two central β strands

Question 3

What is the splicing code for introns

Answer 3

Only 30–40 nucleotides at each end of an intron are necessary for splicing to occur at normal rates – without them splicing will not occur. Here are the indicators that are used to splice intron: - 5ʹ GU (’Splice Donor’)
- 3ʹ AG (‘Splice Acceptor’)
- Branch-point adenosine – usually 20–50 bases from the 3ʹ splice site
- Polypyrimidine tract near the 3ʹ end of the intron – found in most introns
Note: the most important ones are 3’ splice, 5’ splice and polypyrimidine tract. There was a 100% frequency – find GU at 5’ splice and AG and 3’ splice

Question 4

What is exon splicing? Describe the process

Answer 4

Exon splicing – two sequential transesterification reactions

Reaction 1 – Bond between intron 5ʹ phosphorus and exon one 3ʹ oxygen ester bond – exchanged for an intron 5ʹ phosphorus ester bond with the branch-point A residue 2ʹ oxygen

Reaction 2 – The bond between the exon two 5ʹ phosphorus and intron 3ʹ oxygen ester bond – exchanged for an exon two 5ʹ phosphorus ester bond with the 3ʹ oxygen of exon one
o joins the two exons
o releases intron as a lariat structure

Question 5

What are the snRNAs involved in the splicing of pre-mRNAs.

Answer 5

Five U-rich snRNAs – U1, U2, U4, U5, and U6

Question 6

What is necessary for the snRNAs do to in order to splice

Answer 6

Base-pair with pre-mRNA – part of the snRNAs complements part of pre-mRNA – it is important they do or else splicing will not occur (refer to highlighted parts)

Question 7

All eukaryotic mRNAs, except ____ mRNAs, have a 3ʹ ___

Answer 7

1. histone
2. poly A tail

Question 8

Describe the process of the 3’ cleavage and polyadenylation of pre-mRNA

Answer 8

Step 1: CPSF
- CPSF (Cleavage and polyadenylation specificity factor) – binds to the upstream AAUAAA polyadenylation signal
- CStF interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA.
- CFI and CFII binding – stabilize the complex

Step 2: PAP
-PAP (Poly(A) polymerase) – stimulates cleavage at a poly(A) cleavage site (typically 15–30 nucleotides 3ʹ of the upstream polyadenylation signal)

Step 3: Cleavage factors – released
- Downstream RNA cleavage product – rapidly degraded
- PAP – adds ~12 A residues (from ATP) at a slow rate to the 3ʹ-hydroxyl group generated by the cleavage reaction (‘Slow Polyadenylation’)

Step 4: PABPN1
- PABPN1 (Nuclear poly(A)-binding protein) – Binds to the initial short poly(A) tail
- Accelerates the rate of addition by PAP (’Rapid Polyadenylation’)
- Signals PAP to stop adding As after 200–250 A residues have been added

Question 9

Name some things that can yield different mRNAs

Answer 9

Alternative promoters, primary transcript alternative splicing, and cleavage at different poly(A) site cleavages