3rd March - Is the Spliceosome a ribozyme? What is its origin? What is the process in vivo?

Question 1

What is special about group II introns?

Answer 1

They are self splicing

Question 2

Describe the structure of group II introns

Answer 2

They have a similar structure to the spliceosome in complex C:

• Mg2+ ions are in the same place in both active sites
• Both have a bulged U
• Both active sites are the same

Question 3

Why do the group II introns active sites contain Mg2+?

Answer 3

Compensates for the increase in negative charge on these oxygens
They will also catalyse cleavage to some extent

Question 4

How do group II introns spread?

Answer 4

The introns contain all the elements necessary for splicing and often encode reverse transcriptase, thus spread by reverse splicing into RNA

Question 5

Describe the origin of the spliceosome

Answer 5

Believed splicing originated from group II introns
Group II introns can spread by reverse splicing, but it means they lose their splicing ability
Developing their own splicing machinery would mean that they could splice out any mutated introns and would make group II introns redundant

Question 6

Why did the nucleus form?

Answer 6

To prevent the ribosomes translating RNA before it was spliced

Question 7

Is the splicing pathway fixed?

Answer 7

Yes

Question 8

What evidence supports the splicing pathway being fixed?

Answer 8

Depletion of snRNAs in yeast by making the gene repressible with glucose by plasmid shuffling, then performed RT PCR to see if it showed any precipitation of pre-mRNA.

IP vs U1: Yes 
U1 depletion and IP vs U2: No
U1 depletion and IP vs U5: No
--> Need U2 and U1 to bind before U5
U2 depletion with IP vs U1: Yes
U5 depletion with IP vs U1: No
--> U1 -> U2 --> U5

CHiP

• Can be used to measure events in time
• Crosslink with formaldehyde
• Control= Anti-RNAP
• Then Ab against U1, then U2, then U5
• Correlate position of RNAP with event, shows timeline
• -> Showed U1 –> U2 –> U5

Question 9

Outline the evidence that splicing occurs during transcription

Answer 9

Electron Microscopy

• Shows varying lengths of RNA with loops off DNA
• Small lump appears on branches
• Loops correspond to introns
• -> still attached to chromatin but had been spliced

SMI - Coulson 2014

• -Red=Intron
• -Green= 3’ exon
• -Intron was spliced out even when the 3’ exon was still attached to RNAP
• -Confirms that introns can still be spliced out whilst still attached to the gene