18.01.08 Splicing

Question 1

When and where does splicing occur?

Answer 1

In the nucleus pre-mRNA is processed to form mature RNA following 5’ capping, 3’ polyadenylation and splicing.

Question 2

Summarise splicing.

Answer 2

Removal of introns by endonucletolytic cleavage and ligation of exons.

Question 3

How is splicing mediated?

Answer 3

By the major and minor spliceosomes; complexed of snRNA molecules and >100 proteins.

Major snRNAs: U1 U2 U4 U5 and U6
Minor snRNAs: U11 U12, U4atac, U5 and U6atatc

Question 4

What is the GT-AG rule

Answer 4

Introns begin with GT (GU) and end with a AG.

Question 5

What are the sequential steps of pre-mRNA interactions with the snRNA complex?

Answer 5

1. U1 snRNA binds to donor splice site (“GU”) (via it’s complementary sequence: UACUUAC)
2. U2 snRNA binds to branch site (“A” nucleotide), U1 and U2 come together forming a loop in the pre-mRNA
3. U4, U5 & U6 associate with U1-U2 to form the spliceosome; U5 binds both donor & acceptor splice sites
4. Nucleophilic attack of GU (donor) by 2’ hydroxyl group of A of branch point, transesterification #1 (covalent link) forms lariat
5. Cleavage of acceptor (“AG”) transesterification #2 joins exons together
6. Lariat intron & spliceosome unbound