RNA biology lecture 4

Question 1

Transcription + processing

Answer 1

• 3’ end of pre-mRNA needs to be processed
• Occurs by cleavage + polyadenylation
• 2 outcomes = matures 3’ end, instigates transcription termination

Question 2

End of protein coding gene

Answer 2

• Poly A signal in pre-mRNA defines end
• Recognised by components cleavage + polyadenylation complex
• Recognition of polyAsite = co-transcriptional
• Some factors like CPSF = recruited to Pol at promoter via TFIID
• Several factors of complex interact w/ RNAPol II or directly w/ CTD
• Deletion of CTD impairs 3’ end termination

Question 3

What signals direct 3’ end formation

Answer 3

• When RNA pol reaches end of transcription unit, transcribe regions surrounding Poly A site
• Cis elements that direct cleavage = bipartite, AT rich
• 2nd signal ds of cleave site= GT rich
• 2 steps: cleavage + polyadenylation of 5’ product
• Once cis-elements recognised + polyad machinery assembled, cleavage occurs
• 3’OH on maturing mRNA created + 5’ phosphate at pre-mRNA

Question 4

Recognition of poly A site

Answer 4

• CPSF + CTSF
• Once both = assembled, CF1 + polyA polymerase recruited → 3’ end formation complex
• Endonuclease activity in CPSF 73kDa subunit cleaves btw 2 sequence elements

Question 5

5’ 1/2 of cleaved RNA = polyadenylated

Answer 5

• 3’OH = substrate for polyadenylation
• PAP adds 200-250 A, tail covered w/ PABP
• Creates uniform 3’ end, critical for nuclear cytoplasmic export
• PolyA tail assoc w/ PABP w/ CAP binding complex

Question 6

Most but not all mRNAs are polyadenylated

Answer 6

• Replication dependent histone genes x polyadenyl
• x have introns, expression = cell cycle regulated
• Instead of polyA signals have SLBP
• Histone ds element = recognised by U7snRNP through bs in 5’ of U7snRNA
• Leads to cleavage reaction, release mature RNA
• CPSF + Cstf needed

Question 7

Regulation of replication dependent histone gene expression

Answer 7

• Linked to DNA synthesis + cell cycle regulation
• u7snRNA = expressed in cell cycle
• But SLBP = regulated in cell cycle
• SLBP means mRNA is stable in cytoplasm + circularises mRNA by providing interaction point btw CAP + SLBP
• End of mRAN synthesis activates pathway that phosph SLBP → degraded

Question 8

3’ end formation + termination

Answer 8

• Function = no. of polymerases limited, termination means can be regulated, failure to terminate risks affecting ds processes)
• Some cleavage + polyadenyl dissoc from Pol → pre-mRNA
• Cleavage means 1/2 pre-mRNA still attached to Pol which keeps elongating
• Cleavage provides 5’ end that x have cap, degraded

Question 9

Effects of processing on transcription

Answer 9

• pre-mRNA processing can feedback = influence transcription
• Other e.g. of feedback loop e.g. capping = dependent on transcription apparatus but incomplete capping terminates transcription
• Splicing feedback as splicing 1st intron can enhance initiation via TFIIH + splicing U2 ↑ local conc of PTeFb

Question 10

Increasing complexity

Answer 10

• Processing reactions interconnected
• e.g. pre-mRNA not just linked w/ transcription, also communicate w/ each other e.g. CAP binding complex stimulates splicing of 1st intron + 3’ end formation
• e.g. splicing of introns enhances splicing of others