31 - mRNA processing, spliceosome, snrNAs

Question 1

What are the two consensus sequences around 4’ and 3’ splice sites in vertebrate pre-mRNAs?

Answer 1

5’-(splice site)-GU
AG-(splice site)-3’

These are on the intron

Question 2

The branch point is always what nucleotide?

Answer 2

adenosine residue, usually 20-25 residues from the 3’ splice site (on the intron)

Question 3

What are the two steps for mRNA splicing reaction?

Answer 3

1. Cleavage at the 5’ splice site (SS) and joining of the 5’ end of the intro to an A within the intron (the branch point). This reaction yields a lariat like intermediate in which the intron forms a loop
2. Cleavage at the 3’ splice site and simultaneous joining of the exons, resulting in excision of the intron as a lariat-like structure.

Question 4

Splicing of exons in pre-mRNA occurs via two ____ reactions, what are they?

Answer 4

Two transesterification reactions

1. The ester bond between the 5’ P of the intron and 3’ O of exon 1 is exchanged for an ester bond with 2’ O of the branch site A residue
2. The ester bond between the 5’ P of exon 2 and 3’ O of the intron is exchanged for an ester bond with the 3’ O of exon 1, releasing the intron as a lariat structure and joining the two exons.

Question 5

What are small nuclear RNAs (snRNAs)? (4)

Answer 5

• Relatively abundant nuclear RNAs
• Relatively U-rich
• Relatively small
• Exist as ribonucleoprotein particles (RNP)
• Spliceosomal

Question 6

List the five snRNAs and their function.

Answer 6

U1: Binds the 5’ splice site, then the 3’ splice site
U2: Binds the branch site and forms part of the catalytic center
U3: Binds the 5’ splice site
U4: Maska the catalytic activity of U6
U6: Catalyzes splicing

Question 7

What is the proposed mechanism for splicing?

Answer 7

1. U1, U2, U5 and U4/U6 snRNAs, in the form of snRNPs, associate with pre-mRNA in a stepwise fashion to form the spliceosome
2. U1, U2 and U5 recognize and pair with specific sequences in pre-mRNA
3. U1 enters first, then U2
4. U4 and U6 stably pair in a snRNP
5. A U4/U5/U6 triparticle enters
6. U4 is unwound so that U6 can pair with sequences at the 5’ end of U2 to form the catalytically active complex
7. U4 acts as a ferryman to escort U6 to the spliceosome, masking its catalytic domain(s) until spliceosome assembly is complete
8. Then 3’ splice site cleavage occurs

Question 8

What are the two components of a spliceosome catalytic centre?

Answer 8

• U2 snRNA
• U6 snRNA

These are base paired, U2 is also base paired to the branch site of the mRNA precursor

Question 9

Eukaryotic RNA polymerase II CTD interacts with ___ for mRNA processing

Answer 9

• Enzymes that synthesize 5’ cap and with cap binding complex (CBC)
• The 3’ cleavage and polyadenylation enzyme complex
• With spliceosomal components

Question 10

How are different proteins generated from the same pre-mRNA?

Answer 10

Alternative splicing