MBBB Lecture 13 - RNA Processing Flashcards
Three key processing events?
Production of 5’ cap, cleavage and addition of poly(A) tail, 3’ RNA splicing (removal of introns).
Three key enzymes involved in capping?
- Phosphatase - removes phosphate from the original 5’ end.
- Guanyl transferase - adds guanosine base at the 5’ end.
- Methyltransferase - methylates guanosine base.
When does capping occur?
Co-transcriptionally. The enzymes bind to the phosphorylated CTD of RNAP II. When the transcript emerges, the enzymes begin to modify the 5’ cap.
What does the poly(A)tail consist of?
Roughly 200 adenosine residues.
What consensus sequences are involved in cutting the transcript for polyadenylation?
Consensus sequences = hexamer AAUAA, GU-rich element. In between is a CA dinucleotide.
Key proteins that bind to the consensus sequences before polyadenylation?
- Cleavage stimulation factor F (CstF), binds GU rich region.
- Cleavage and polyadenylation specificity factor (CPSF), binds AAUAA hexamer.
The proteins involved in binding to the consensus sequences of polyadenylation are bound to and travel with RNAP II at the CTD. Why?
They are deposited at the 3’ end of the RNA molecule once it has been transcribed. They bind to each other, forming a loop. CA dinucleotide is presented to an endonuclease.
Which enzyme adds 200 A nucleotides to the 3’ end?
Poly-A-polymerase (PAP).
Why add a 5’ and a 3’ polyA tail? Why do we need these modifications?
The 5’ cap and 3’ poly(A) tail have stabilising effects. Eukaryotic mRNAs are much more stable because they must be transported to the cytoplasm.
RNA splicing - what are the three sequences that signal splicing should occur?
5’ splice site = generally a GU.
Branchpoint = A.
3’ splice site = AG often preceded by a run of pyrimidines.
Transesterification reactions involved in splicing?
Branchpoint A has a 2’ OH that attacks the backbone of the 5’ SS (GU), releasing the 1st exon. Loop structure called the lariat.
Next step is another nucleophilic attack at the 3’ splice site.
What does the spliceosome consist of?
Small nuclear ribonucleoprotein particles (snRNPs). snRNPs are composed of 5 U RNAs.
Steps of splicing?
- U1snRNP binds to the 5’ splice site and U2snRNP binds the branchpoint.
- The U1snRNA pairs with the pre-mRNA at the 5’ SS. U2snRNA pairs at the branchpoint. This is not perfect - the branchpoint A sticks out ready for nucleophilic attack.
- U4/U6 displaces U1; U5 binds the 5’ exon. U6 interacts with U2.
- Two step trans-esterification reaction,
Auxillary factors bind to the pyrimidine sequence.
The splice sites of consensus sequences are not well conserved. What helps their identification?
Within mRNA there are exonic splicing enhancers (ESEs) = sequences within exons that interact with SR proteins.
SR protein = rich in serine and arginine. Their binding helps stabilise the snRNPs and recruit them to the 5’/3’ splice sites.
Why is alternative splicing used?
It is a mechanism that increases the protein coding capacity of higher eukaryotes.
