RNA Processing in Eukaryotes - Lecture 13

Question 1

What are the three RNA processing reactions?

Answer 1

○ Capping
○ Polyadenylation
○ Splicing

Question 2

How does mRNA capping work?

Answer 2

○ One phosphate group is lost from the 5’ end
○ 5’ to 5’ linkage
○ Methyl group added to guanosine
○ Methyl group added to first 2 nucleotides at the hydroxyl group

Question 3

Why is mRNA capping important?

Answer 3

○ Linked to transcription initiation
○ Increases stability of mRNA
○ Required for efficient splicing
○Nuclear export
○ Translation initiation

Question 4

How does polyadenylation work?

Answer 4

○ Signals for cleavage/polyadenylation (AAUAA, G/U or U region)
○ CPSF binds to AAUAA
○ CstF binds to GU and recruits cleavage factors and polyA polymerase
○ Cleavage and addition of polyA (lots of As added)

Question 5

Why is polyadenylation important?

Answer 5

○ PolyA tailing functionally linked to transcription termination
○ Major role in stability and translation efficiency
○Length of tail is proportional to RNA stability

Question 6

What are the signals for splicing?

Answer 6

○ 5’ splice (donor) site: Purine (U)
○ Branchpoint: Any nucleotide
○ 3’ splice (acceptor) site: Pyrimidine (Y)

Question 7

What are the steps for splicing?

Answer 7

Step 1:
○ Cleavage at 5’ splice site and lariat formation at branchpoint sequence
○ 5’ phosphate joins to 2’ hydroxyl group to form loop structure
Step 2:
○ Cleavage at 3’ splice site and removal of intro region and exon ligation
○ Exons join together by esterification

Question 8

What carries out splicing?

Answer 8

snRNP’s and protein splicing factors

Question 9

What is a snRNP and which ones are involved?

Answer 9

○ Small nuclear RNA + numerous proteins
○ Involved: U1, U2, U4, U5, U6

Question 10

How do snRNP’s carry out splicing?

Answer 10

○ U1 finds intron/exon boundary at donor site
○ U2 recognises branchpoint and binds to it
○ A complex of U4, U5, U6 displace U1
○ Active site of splicesome includes U2, U5, U6 and loops out intron

Question 11

What is alternative splicing?

Answer 11

Exon inclusion/exclusion: proteins with altered sequence or truncated if included exon has a stop codon

Question 12

What are examples of exon inclusion/exclusion?

Answer 12

○ Sex determination in fruit flies
○ Control of flowering time
○ Olfactory receptor diversity

Question 13

What are isoforms?

Answer 13

○ Different proteins can be produced from a single gene
○ Isoforms may have different functions or no function at all