20th Feb - Splicing, In Vitro and the chemical reactions

Question 1

What are the two major problems for establishing splicing reactions in vitro?

Answer 1

Getting a defined piece of RNA

Adding the RNA splicing system

Question 2

What was the solution to producing a defined piece of RNA?

Answer 2

Using T7 RNAP

Question 3

What was the solution to getting the RNA splicing system in vitro?

Answer 3

Injecting Xenopus oocyte nuclei however this was of limited use biochemically
Now use cell extracts commonly from HeLa cells then put them into a lower ionic strength buffer

Question 4

How is a nuclear extract made?

Answer 4

By putting nuclei in hyperosmotic medium, causing competition between the proteins in the nucleus, causing all nuclear proteins to leave the nucleus except histones

Question 5

Outline Krainer’s experiment which showed that mRNA is made as part of a 2-step reaction

Answer 5

Took out samples at varying times and analysed the products on a gel
Found that the pre-mRNA yield declines with time
Found that the mRNA yield increases with time but at a different rate

Question 6

What led to the hypothesis that the intermediates of splicing were branched or circular?

Answer 6

They ran differently on different strength gels, they appeared to gain Mw

Question 7

How was it identified that splicing intermediates were lariats?

Answer 7

RT PCR with 2 primers

• • Circular would lead to one band on the gel
• • linear would lead to two different weight bands
• -> Primer 1 strand was very short

Thus could it be branched? Lariat or Y-shaped?

• -Cut with RNase H, which cuts where an oligo is branched
• • Y shaped would create 2 molecules
• • Lariat would create 1 molecule
• -> 1 molecule therefore the intermediate was a lariat

Question 8

How did was the branch site mapped to within a few NTs?

Answer 8

1. Used RNase H
2. Added NT’s at various points along the intron and mapped the linear piece
3. Cleavage within the loop would produce an aberrantly moving Y shaped molecule –> huge shift in mobility. When cleave at the branch point –> a straight full intron
4. From this they used the specificity of ribonucealses to show the branch point, using labelled GTP

–> Cut with T1 and T2 gave two radioactive G and 1 radioactive A

Question 9

How was the branch site identified to 1 NT?

Answer 9

RNase T2 digests all RNA to Np except branchpoints which end up as X (pYp)(pZp) (where X is the branchpoint and Y is the linked base and Z is the base after)
A phosphatase can then be applied to remove the end phosphate

1. RNA was labelled with individually radio-tagged NTPs
   - -[alpha32P] ATP –> no labelling
   - -[alpha32P] CTP –> labelling
   - -[alpha32P] GTP –> labelling
   - -[alpha32P] UTP –> no labelling
2. Y and Z = G and C
3. Thus the branch is A

Question 10

How was it identified that step 1 of splicing was a transesterification?

Answer 10

Replaced 1 oxygen with sulphur making the phosphate group chiral.
A transesterification would cause an umbrella conversion