Lecture 13

Question 1

Steps required to produce functional mRNA from precursor mRNA

Answer 1

• Addition of 5’ cap (capping)
• Addition of 3’ tail (polyadenylation)
• Spicing (usually)
• Editing (sometimes)

Question 2

RNA cleavage

Answer 2

rRNA and tRNA synthesized as long precursor RNA molecules

Processed to correct length by ribonucleases

Ensures RNAs available in same amounts in cell

Question 3

Cap structure at 5’ end of eukaryotic mRNA

Answer 3

• Guanine nucleotide added to 5’ end of mRNA via 5’-5’ linkage
• Guanine methylated in N7 position
• 2’-O position of 2nd and sometimes 3rd ribose also methylated in higher eukaryotes

Question 4

What does the 5’ cap do?

Answer 4

• Protects mRNA from degrading
• Translatability - cap stimulates translation of mRNA ~300 fold
• Transport from nucleus to cytoplasm

Question 5

Capping involves 3 steps

Answer 5

1. RNA triphosphatase removes terminal phosphate at 5’ end
2. Guanylyl transferase uses GTP to attach GMP
3. Guanine is methylated by methyltransferase

Question 6

When does capping occur

Answer 6

After first 20-30 nucleotides have been synthesized

Question 7

Polyadenylation

Answer 7

• mRNA cleaved immediately after a CA between AAUAAA hexameric sequence and a U or GU rich sequence
• 3’ end has adenosines added by poly(A)polymerase

Question 8

Splicing

Answer 8

• precursor mRNA often contains introns for mature mRNA template
• Removed by transesterification reactions
• Most removed by spliceosomes in metazoa
• Spliceosome contains proteins and snRNAs

Question 9

What are intron boundaries defined by?

Answer 9

Presence of 5’-GU and 3’-AG

Question 10

What are intron boundaries recognised by?

Answer 10

Spliceosome (formed from 5 small nuclear riboproteins or snRNPs, eahc containing 100-300nt of snRNA and proteins)

Question 11

EJC complex

Answer 11

• Left at splice junctions after splicing
• EJC marks transcription during processing and interacts with export and translation proteins
• Prevents incompletely processed RNA from being exported

Question 12

How does RNA pol II coordinate capping, splicing and polyadenylation?

Answer 12

• Repeated amino acid sequences in RNA pol CTD become partially phosphorylated
• Phosphorylayed CTD recruits enzymes that add 5’ cap to growing RNA
• RNA pol continues elongation,
• Addition phosphorylation recruits complex that cleaves and polyadenylates 3’ end of mRNA

Question 13

RNA editing

Answer 13

• Adds/deletes bases from pre-mRNA
• Chemically alters bases that don’t match DNA

Question 14

Substitution editing

Answer 14

APOB gene contains 29 exons
- exons have 4564 codons
- Codon 2153 is CAA - glutamine

Liver cells - Apolipoprotein B-100 cotains 4563 amino acids

Intestinal cells - C nucleotide deaminated to U in codon 2153 (CAA -> UAA)
Translation stops at this codon forming apolipoprotein B-48 with 2152 amino acids

Question 15

What determines RNA stability

Answer 15

Poly-A tail and 5’ cap structure

Question 16

What controls RNA degradation and how?

Answer 16

AREs or AU rich elements- found in 3’UTR

Found in mRNAs that are required for a very short period of time

Question 17

mRNA degradation - RNAi

Answer 17

2 types of small RNAs (sRNAs) that allow degredation

• siRNAs (Small interfering RNAs)
• Derived from processing of longer dsRNAs
• miRNAs (micro RNAs)
• Derived from RNA pol II transcripts

Question 18

Production of miRNAs and siRNAs

Answer 18

miRNA or siRNA suplex loaded onto Argonaute protein

One strand degraded - forms mature miRISC or snRISC

Question 19

Silence genes of miRISC

Answer 19

• Binds 3’UTR mRNA - do not directly cleave RNA - not fully homologous
• Repress translation
• Activate adenylation, decapping and exonucleolytic degradation

Question 20

siRISCs silence genes