Regulation of RNA Processing in Eukaryotes

Question 1

What does transcription in eukaryotes produce?

Answer 1

Immature, pre-mRNA

Question 2

What are the three processes pre-mRNA undergoes after transcription?

Answer 2

5’ capping
splicing
3’ poly-adenylation

Question 3

What indicates that 5’ capping can begin?

Answer 3

Phosphorylation of Ser5

Question 4

What indicates that splicing can begin?

Answer 4

Ser2 is also phosphorylated (after Ser5)

Question 5

What indicates that the Poly-A tail can start getting added?

Answer 5

Ser5 is dephosphorylated while Ser2 remains phosphorylated.

Question 6

What is a kinase?

Answer 6

A protein that can add a phosphate to anything.

Question 7

What is a phosphotase?

Answer 7

An enzyme that removes phosphate.

Question 8

Explain the process of 5’ capping.

Answer 8

1. γ phosphate of transcript is removed from 5’ end
2. β and γ phosphates removed from GTP
3. 5’-5’ phosphotriester links GMP and transcript head-on
4. Methyl group added to N7 position on guanine
5. A cap binding protein (CBP) binds to 5’ cap

Question 9

What nucleotide is the branch point for splicing in eukaryotic transcript processing?

Answer 9

A

Question 10

Explain the process of splicing in eukaryotic transcript processing.

Answer 10

1. A uses 2’ OH to attack phosphate connecting 3’ end of exon-1 and 5’ end of intron
2. Phosphate attaches to 2’ C of A
3. 5’ end of exon 2 gets re-attached to 3’ end of exon 1

Question 11

What are the conserved nucleotides that indicate the beginning and end of an intron?

Answer 11

5’ end: GU
3’ end: AG

Question 12

What is a spliceosome?

Answer 12

• Identifies Exon / Intron Junctions & Excises Introns- Splices Exons
• Forms during transcription

Question 13

What type of RNA plays a major role in splicing?

Answer 13

Small nuclear RNA (snRNA)

Question 14

What are the major units of a spliceosome made of?

Answer 14

Protein + snRNA = snRNP

Question 15

Identify the names of the protein and snRNPs in the splicosomes.

Answer 15

*BBP branch point binding protein
*snRNP U2
*snRNP U1
*snRNP U6
*snRNP U4
*snRNP U5

Question 16

Explain how splicing occurs specifically with the spliceosome (4 parts)

Answer 16

1. U1 snRNP binds 5’ splice junction and BBP + U2AF (U2
   auxiliary factor) binds to branch point
2. U2 snRNP displaces BBP + U2AF and binds to branch
   point + 3’ splice junction
   * Notice that U1 and U2 interaction vs. RNA is
   mediated by RNA-RNA hybridization
3. U4/U6 and U5 joins the complex, followed by U4
   dissociation. This causes conformational change to
   open the catalytic site for first phosphoryl-transferase
   reaction (Adenine attacking with free OH)
4. Second phosphoryl-transferase reaction occurs,
   joining the two exons together

Question 17

What is alternative splicing? What is its advantgae?

Answer 17

• Exons can be joined in different
  patterns
• Increases the number of proteins
  encoded by a single gene

Question 18

List the nucleotide sequence of the signal for trimming and poly-A addition.

Answer 18

AAUAAA

Question 19

What proteins recognize the poly-A sequence?

Answer 19

CstF and CPSF Cleavage stimulating factor / cleavage and polyadenylation specificity factor

Question 20

How many A residues are added for the Poly-A tail? What adds them?

Answer 20

250; Poly-A polymerase (PAP)