RNA processing: 5' capping, splicing and 3' end modifications (lect 1 + 2)

Question 1

Order of gene expression events

Answer 1

transcription
5’ capping of RNA
RNA splicing and polyadenylation
RNA export
translation

Question 2

What is the function of the 5’ cap?

m7G cap

Answer 2

-protects RNA from degradation in nucleus
-stabilises mRNA
-ensures mRNA gets exported and translated

Question 3

What occurs during capping?

Answer 3

-addition of guanosine
-guanosine methylated (position 7)

Question 4

When does capping occur?

Answer 4

co-transcriptionally

Question 5

What occurs during splicing?

Answer 5

-U1 recognises 5’ splice site.
-triple snRNP (U4, 5 and 6) recruitment causes structural rearrangements
-U1 and 4 leave, leaving catalytic spliceosome and lariat structure
-3’ splice site cleaved and exons joined (lariat containing intron released and degraded)

Question 6

What occurs during 3’ end formation?

Answer 6

-mRNA cleaved at CA site by protein complex (involving cleavage factors)
-GU/U-rich sequence degraded in nucleus
-remaining mRNA polyadenylated at CA-OH by diff protein complex (involving PABP)

Question 7

What is the Pol2 CTD?

Answer 7

carboxy terminal domain
-largest subunit of Pol2
-made of repeated aa seq YSPTSPS (these are dynamically modified, eg. phosphorylated during transcription to allow factors to be recruited)
-essential for splicing and 3’ end processing

Question 8

What does the phosphorylation of Ser5 on the Pol2 CTD drive?

Answer 8

-recruitment of capping enzyme to promote methylation of cap
-spliceosome assembly

Question 9

What does the phosphorylation of Ser2 on the Pol2 CTD drive?

Answer 9

recruitment of CstF (cleavage factor) for poly(A) site cleavage

Question 10

What is the purpose of alternative splicing?

Answer 10

diversify mRNAs

Question 11

Examples of alternative splicing

Answer 11

-cassette exon (either included or skipped)
-mutually exclusive exons
-introns retained
-alternative splice sites (3’ or 5’)
-alternative promotors (diff transcription start sites)
-alternative polyadenylation sites (depending where’s spliced)

Question 12

What factors influence splicing?

Answer 12

-Exon bound SR proteins (enhance splice sites to influence U1 recruitment and exon definition)
-Silencer proteins (eg. hnRNPs, repress splice sites by binding to whole intron or silencer seqs)

Question 13

Sex determination in Drosophila controlled by alternative splicing

Answer 13

Males: include exon 3, so no sex lethal protein (Sxl) made
Females: skip poison exon 3, so produce Sxl

Question 14

Examples of scenarios controlled by alternative splicing

Answer 14

-Drosophila sex determination (exon 3 included in females so Sxl made, not in males)
-tropomyosin (alt splicing is cell-specific, meaning diff forms of tropomyosin in diff muscle types)

Question 15

What causes Duchene muscular dystrophy?

Answer 15

Mistakes in splicing of dystrophin gene (dystrophin is a long gene so easy to have splicing errors)
-out of frame transcripts make dystrophin truncated

Question 16

How does speed of transcription effect splicing patterns?

Answer 16

high elongation rate RNAP will skip exon if the 3’ splice site is weak (will include exon is 3’ ss strong, or if low elongation rate)

Question 17

Example of alternative polyadenylation

Answer 17

IgM mRNA has an early, weak cleavage/polyA site (encodes soluble, secreted form mRNA for plasma cells) and a late, stronger polyA site (encodes membrane-bound form mRNA for B cells)
-CstF64 levels control which form selected for
-high CstF64 selects for the weak site, low levels selects the stronger site

Question 18

Link between UTR length to cancer

Answer 18

cancer cells have shorter UTRs
-because they have early polyA sites
-makes it easier to evade RNAi

Question 19

Link between UTR length and mRNA stability

Answer 19

longer UTRs have more miRNA binding sites so are less stable (more miRNAs can bind for RNAi)

Question 20

How do U1 snRNA levels effect mRNA length?

Answer 20

U1 represses early polyA sites, so that CstF doesn’t recognise them to ensure the desired mRNAs are obtained (telescripting)

-higher levels of U1 compared to other snRNAs indicated that it doesn’t only function in spliceosome assembly