Module 4 Section 2

Question 1

Processing of RNA Euk vs. Prok

Answer 1

• mRNA subject to constant degradation
• In prokaryotes, RNA translated before transcription is terminated
• In Euk, newly transcribed pre-mRNA subject to modifications prior to exit from nucleus

Question 2

If eukaryotic pre-mRNAs were not post-transcriptionally modified, what would be the consequence?

Answer 2

• enhanced degradation (end caps)
• Impaired ribosome binding
• Translation of non-coding DNA sequences

Question 3

Processing of pre-mRNA

Answer 3

• 5’ capping
• 3’ polyadenylation
• splicing
• nucleotide editing

Question 4

5’ Capping

Answer 4

• 7-methyl guanosine attached to mRNA through 5’-5’-triphosphate linkage
• mediated by guanylyltransferase
• additional methyl groups often added to 2’OH of ribose sugars at the 1st and 2nd nucleotides adjacent to cap, and to N7 on the guanine (the cap one)
• occurs early in transcription (after addition of 20-30 nucleotides)
• Mediated by C-term domain (CTD) of RPBI
• Phosphorylation of CTD recruits specific RNA modifying enzymes (including guanylyltransferase)
• Cap-binding complex (CBC) recruits mRNA to ribosome

Question 5

Purpose of 5’ cap

Answer 5

• prevents recognition of the 5’ end by exoribonucleases

- mediates binding of mRNA to the ribosome

Question 6

3’ polyadenylation steps

Answer 6

1. Pol II transcribes the poly(A) addition site at term end of transcript
   Marked by two sequence elements:
   -highly conserved sequence 5’-AAUAAA-3’ 10-30 nucleotides upstream of cleavage site
   -GT rich region 20-40 nucleotides downstream of cut site
2. Polyadenylation factors bind poly(A) signal upstream of cleavage site
   -cleavage generates free 3’OH that defines the end of mRNA
3. “A” residues are added to free 3’OH immediately in Poly(A) polymerase (PAP) catalyzed rxn
   -A-residues are bound by Poly(A) binding proteins (PABP) which protect 3’mRNA from exoribonucleases

Question 7

mRNA circularization

Answer 7

• 5’ cap and 3’ polyA tail associate with subunits of elF4F to circularize mRNA
  1. mediates ribosome binding
  2. promotes translational efficiency
  3. ensures complete processing

Question 8

Splicing of Pre-mRNA

Answer 8

-exons: typically <1000 nucleotides
Introns:
-50-20000 nucleotides
-do not code for protein
-contain regulatory sequences
-allow for alternative splicing of genes
5'UTR and 3'UTR are NOT spliced out

Question 9

Splicing

Answer 9

• the controlled act of breaking and joining specific phosphodiester bonds to excise introns
• cannot reorder RNA (ie. cannot have 2, 1, 3)

Question 10

Spliceosome

Answer 10

• mediates splicing
• complex of 5 small nuclear ribonuclear proteins (snRNPs)
• snRNP = snRNA and protein subunits

Question 11

Reverse transcription

Answer 11

• generation of cDNA
• make use of 3’polyA to make sure only mRNA is replicated
• use polyT sequence as a primer (Oligo dT primer)