8th March - The impact of transcription on splicing efficiency and splice site selection

Question 1

Outline an experiment that measured the rate of splicing in vitro

Answer 1

Applied DRB to inhibit new transcription (by inhibiting P-TEFb therefore stopped at first pause site) and allow all existing transcription to be fully spliced.

Wash out DRB therefore restarting transcription

Assayed by RT PCR to measure the levels of spliced and unspliced RNA
-Showed splicing takes 5-10 minutes

THIS IN VITRO TIME IS VERY LONG, IT IS FASTER IN VIVO

Question 2

Outline an experiment that measured the rate of splicing in vivo

Answer 2

Caulon 2014 - SMI
Spliced RNA was Red and Green
Spliced RNA was Green
Used modified RNA
-->RNAP mapping doesn't see pausing around the 3'ss
-->Splicing only took 20-30s

Question 3

Why is splicing in vivo faster than in vitro (2 models)?

Answer 3

1. When RNAP rolls along DNA it holds the 5’end of the intron near the exit tunnel so that when the 3’ss leaves RNAp splicing can begin immediately. This can be tested by putting a self cleaving ribozyme sequence into the intron. If the 5’ss is brought close to the RNAP then nothing will happen, if its not it will drift off
2. The phosphorylated CTD recruits splicing complex components therefore they are not needed for diffusion. The CTD recruits U2F65 via S2 phosphorylation.

Question 4

Outline an experiment that supports the phosphorylated CTD model of why splicing is faster in vivo than in vitro

Answer 4

Gu 2013

Mutated ser2 to alanine and found a substantial reduction in splicing ability

Question 5

What is the first come first served model?

Answer 5

The concept that the 5’ss will use the next 3’ss as another one hasn’t been synthesised yet as splicing occurs as transcription is still taking place.

Question 6

How could nucleosomes affect splicing?

Answer 6

It could affect the rate of transcription - the speed humop model, therefore promoting splicing of weak splice sites
It could recruit splicing factors

Question 7

How do introns affect the level of expression?

Answer 7

Believed that the sequence of the exon plays a role in the transition from pausing to elongation

When paused RNAPII has a large complex which forms a bundle around 7SKRNA.
SRSF2 leaves 7SKRNA and binds to the ESE pulling cyclin T and cdk9 off 7SKRNA causing elongation

Question 8

How does U1 affect the direction of transcription binding?

Answer 8

Believed that binding sites for U1snRNPs bind U1 protecting the RNA from cleavage and polyadenylation for the next 1000nt
In the other direction there is a surplus of polyA sites and a lack of exon sites