19.01.08 splicing

Question 1

What does splicing do

Answer 1

Removes introns by endonucleolytic cleavage and ligation of exons

Question 2

What is the start end sequence of most introns

Answer 2

Start- GT (splice donor), end- AG (splice acceptor).

Question 3

What is the branch sequence

Answer 3

Located 20-50 bases upstream of acceptor site.

Question 4

Consensus sequence of branch site

Answer 4

CT(A/G)A(C/T). The A is critical in enzymatic reaction

Question 5

How any snRNAs involved in splicing

Answer 5

5

Question 6

Steps of splicing

Answer 6

1. Endonucleolytic cleavage at 5’ end of intron (donor site)
2. Nucleophilic attack. GU sequence forms “lariat” loop with A at branch point.
3. Endonucleolytic cleavage at 3’ end (acceptor site).
4. Exons ligated together and intron degraded.

Question 7

How many spliceosomes are there

Answer 7

2, major and minor spliceosome

Question 8

What is the major spliceosome composed of

Answer 8

U1, U2, U4, U5 and U6 small nuclear RNA molecules. Plus other proteins

Question 9

What is the minor spliceosome composed of

Answer 9

U11, U12, U4atac, U5, U6atac. Plus other proteins

Question 10

What is the first step of splicing

Answer 10

U1 snRNA binds to donor splice site(GU)

Question 11

Where does U2 snRNA bind

Answer 11

Branch site A nucleotide. U1 and U2 then come together to form a loop in pre-mRNA.

Question 12

What binds to U1 and U2 to form spliceosome

Answer 12

U4, U5, U6.

Question 13

Nucleophilic attack of GU (donor) is caused by what

Answer 13

2’ hydroxyl group of A branch point.

Question 14

What bonds the G from donor with A of branch site

Answer 14

transesterification, in which a hydroxyl (OH) group on a carbon atom of the adenine “attacks” the bond of the guanine nucleotide at the splice site. The guanine residue is thus cleaved from the RNA strand and forms a new bond with the adenine.

Question 15

What happens during the second transesterification reaction

Answer 15

WHhen acceptor site is cleaved the OH group at 3’ end of exon attacks phosphodiester bond at 3’ splice site. The lariat intron and spliceosome are then released.

Question 16

Is energy required for transesterification

Answer 16

No. Energy neutral. ATP is, however, needed to arrange spliceosome

Question 17

What regulates splicing

Answer 17

Efficacy of consensus sites and trans-acting/ cis-acting elements.

Question 18

What is an exonic splicing silencer (ESS)

Answer 18

Short region of an exon that is cis-regulatory unit. Inhibit or silence splicing of pre-mRNA. Promote exon skipping.

Question 19

What is an exonic splicing enhancer (ESE)

Answer 19

DNA sequence motif that enhances accurate splicing of heterogenous nuclear RNA (hnRNA)/pre-mRNA into mRNA.

Question 20

Mutations in ESE motifs

Answer 20

Some point mtuations in ESEs can inhibit affinity for splicing factors and alter alternative splicing. Leading to altered mRNA and protein translation.

Question 21

What percentage of human genes are alternatively spliced

Answer 21

92-97%

Question 22

What is the purpose of alternative splicing

Answer 22

To regulate gene expression. Major contributor of proteomic diversity.

Question 23

Where does 80% of splicing variability take place

Answer 23

ORFs. Alters translated proteins

Question 24

Where does 20% of splicing variability take place

Answer 24

UTRs. Affect mRNA processing, stability and localisation