Lecture Notes

Week 6 Notes: RNA splicing & processing

Introduction
 pre-mRNA: nuclear transcript that is processed by modification and splicing to form mRNA
 RNA splicing: excision of introns from RNA and connecting the exons into a continuous mRNA
 RNA modification in the nucleus by additions to the 5’ and 3’ ends and by splicing to remove introns
o Poly(A) modification (3’ end) is intrinsically linked to transcriptional termination

Nucleotides-differences b/w DNA and RNA Synthesis from 5’-3’

Pol II CTD phosphorylation- The CTD code
 Dynamic phosphorylation of RNA pol II CTD repeats is coupled to transcription and acts as a code that
controls mRNA synthesis/processing

Processing: RNA capping at 5’ end
 Guanosine: guanine connected to mRNAvia 5’ to 5’ triphosphate linkage
o Guanosine has been methylated by methyltransferase
 Capping occurs during transcription and is important for release from pausing of transcription
 3 forms of capping: all contain cap 0. The 5’ cap of most mRNA is monomethylated, but some small
noncoding RNAs are trimethylated
o 3 enzymatic activities required to add the cap:
 RNA triphosphatase (H2O  H2PO4),
 Guanylyltransferase (GTP  PPi) (both enzymes present in same capping enzyme)
 N7 G-methyl transferase (S-adenosylmethionine  S-adenosylthomocysteine)

Capping enzyme recruited by CTD of RNA pol II
 RNA pol II CTD is phosphorylated on Ser-5 to target a transcript for capping
 CE interacts with Ser-5 phosphorylated pol II

Transcription termination involves 3 steps
1. Cleavage at poly(A) site
2. Addition of poly(A) tail at new 3’ end
3. Transcription termination downstream from cleavage site

3’ ends of mRNAs generated by cleavage and polyadenylation
 The sequence AAUAAA is a signal for cleavage to generate a 3’ end of mRNA that is polyadenylated
o Reaction requires protein complex that contains specificity factor, endonuclease & poly(A)
polymerase
 Specificity factor and endonuclease cleave RNA downstream of AAUAAA

, Week 6 Notes: RNA splicing & processing

 The specificity factor and poly(A) polymerase add 200 AA residues processively to 3’ end
o Poly(A) tail controls mRNA stability and influences translation
o Cytoplasmic polyadenylation plays a role in Xenopus embryonic development

Machinery required for cleavage only
 CstF: binds to GU-rich sequence
 CFI & CFII: little known about their function
 CTD: binds both CPSF & CSTF

Machinery required for polyadenylation only
 PAB II interacts with CPSF, stimulating rate of poly(A) addition and controls poly(A) tail length

Machinery required for both cleavage and polyadenylation
 CPSF: binds to poly(A) signal AAUAAA
 PAP: adds poly(A) tail and is recruited by CPSF
 Symplekin: part of larger complex that includes CstF & CPSF
o Helps to assemble/stabilise the CstF complex and hold the polyadenylation machinery together

3’ end of mRNA formation of Histone mRNA requires U7 snRNA
 Histone mRNA expression is replication dependent and regulated during cell cycle
 Histone mRNAs are not polyadenylated: their 3’ ends are generated by cleavage reaction that depends on a
conserved hairpin structure in mRNA

o Cleavage reaction: SLBP (stem-loop binding prot) binds SL (stem-loop) structure
U7 snRNA pairs with adjacent single-stranded region
 Catalysed by a factor shared with the polyadenylation complex

3’ end mRNA processing is critical for transcription termination
Pol II: Transcription can be ended in various ways by different RNA polymerases . mRNA 3’ end formation signals
termination of pol II transcription

 Elongation complex changes conformation upon recognising poly(A) site
 After cleavage a splicing factor, SF, recruits Xm2 which digests downstream RNA until it reaches RNA pol II
o Helicase helps release pol II

Pol I and II: use specific terminators to end transcription
 Pol I: 2 discrete termination sits are recognised by a DNA-binding protein (TTF1 in mouse) and cleavage
mediated by endonuclease Rnt1
 Pol III: defined terminator sequence in DNA molecule (oligo dT) signals release of RNA pol

RNA splicing
 Excising introns from RNA and connecting exons into a continuous mRNA
 Occurs during and/or after transcription, and mRNA is exported

Nuclear splice sites are short sequences
 Splice sites: sequences immediately surrounding the exon-intron boundaries
o 5’ splice site at the 5’ (left) end of intron includes consensus sequence GU
o 3’ splice site at the 3’ (right) end of intron includes consensus sequence AG
o GU-AG or U2-type introns (98% of human introns)

o 5’ splice site at the 5’ (left) end of intron includes consensus sequence AU
o 3’ splice site at the 3’ (right) end of intron includes consensus sequence AC
o AU-AC or U12-type introns (>1% of human introns)