Lecture 10: Pre-mRNA Processing in Eukaryotes

Question 1

What is capping and where does it take place?

Answer 1

1. Addition of cap structure at 5’ end
2. Linked to 5’ mRNA by 5’-5’
   triphosphate bridge
3. Enzymes required:
   a. 5’ triphosphatase
   b. guanylyl transferase
   c. 7-methyl transferase
4. Once cap structure made, will be bound
   to by “cap-binding complex”
   a. gives ability to leave cell
   b. and take part in translation
   c. promote correct splicing

Question 2

What is polyadenylation and where does it take place?

Answer 2

1. 3’ end
2. poly-A tail
3. Anything transcribed past the poly-A
   site is degraded
4. Can be used for mRNA purification
5. mechanism
   a. poly-A signal; poly-A cut site down
   stream
   b. 3’ processing complex binds and
   cleaves
   c. ~10 A’s added (potentially hundreds)

Question 3

How is splice sites recognised?

Answer 3

1. 5’ = Splice junction donor
   a. just inside intron
2. 3’ = Spice junction acceptor
   a. just outside intron
3. ONLY ONE 100% CONSERVED
   CONSENSUS SEQUENCE…
   a. DNA = GT… …AG
   b. RNA = GU… …AG
   c. branch point = A residue

Question 4

How does splicing take place?

Answer 4

1. “Lariat Formation”
2. attack of the “branch point” 2’ A-residue to the splice junction donor (5’)
3. OH on 3’ of exon attacks 5’ mRNA exon
4. exposes an OH group of the lariat loop
   which gets degraded

Question 5

What is responsible for splicing?

Answer 5

1. Spliceosome - five U-rich small nuclear 
   RNAs (snRNAs) designated:
   a. U1
   b. U2
   c. U4
   d. U5
   e. U6
2. (Form complexes with 6-10 proteins 
    each)
3. U2 pairs in a way that the 'A branch 
    point' sticks out -allows first 
    transesterification reaction

Question 6

What is the order of events of splicing?

Answer 6

1. U1 and U2 bind

2. U4, U5, and U6

Question 7

What is alternative splicing

Answer 7

1. Ability of a single pre-mRNA to form
   different products
   a. 35-59% of human genes show
   variably spliced products

Question 8

How is alternative splicing regulated?

Answer 8

1. Negative factors: prevent U1 or U2
   binding OR block spliceosome assembly
   after U1/U2
2. Positive factors: enhance spliceosome
   assembly at sites that are otherwise
   poorly recognised

Question 9

What is the role of RNAP2 C-terminal domain?

Answer 9

1. Regulates timing of splicing, capping, 
   poly-adenylation 
2. 52 repeats of YSPTSPS
3. Pol 2A unphosphorylated = initiation
4. Pol 2O phosphorylated = elongation

5. Capping = Phosphorylation of serine 
    5; capping enzyme binding 
6. Splicing = phosphorylation of serine 2; 
    splicing factors bind
7. Poly A formation

Question 10

How does mRNA export take place?

Answer 10

1. Nuclear exporter:
   a. heterodimeric RNA binding protein
   b. associates with the FG (phenylalanine-
   glycine) repeats of nucleoporin
   proteins that line nuclear pore
   c. mRNA exporter recycles back to
   nucleus

Question 11

How and why does mRNA maintain stability

Answer 11

1. 5’ cap:
   a. aids mRNA nuclear export
   b. protects 5’ - 3’ exonuclease
   degradation in cytoplasm
   c. enhances transateability
2. Poly-A tail
   a. all of above
3. 3’UTR:
   a. contains regulatory elements
4. VAST VARIATION ON STABILITY
   a. <30 mins to tens of hours
   b. short lived often regulatory proteins
   c. long lived often structural/metabolic
   enzymes

Question 12

How are mRNAs degraded?

Answer 12

1. many short love mRNAs contain AU-rich elements in 3’UTRs
2. AUUUA elements can bind specific proteins that interact with the deadenylating enzyme and the exosome, promoting rapid mRNA turnover

Question 13

What are the 3 pathways for mRNA degradation?

Answer 13

1. Decapping pathway (deadenylation- 
   independent)
   a. decapping
   b. exonucleolytic activity
2. Deadenylation-dependent
   a. Poly-A shortening 
   b. decapping OR exonucleolytic decay
   c. if dacapped, then exonucleolytic decay
3. Endonucleolytic pathway
   a. endonucleolytic cleavage
   b. exonucleolytic decay