Lecture 19 - What happens to RNA between initiation of transcription and translation? Flashcards
How much of the total RNA produced by a cell is mRNA/ funcitonal RNA?
mRNA
-2% of total
Functional RNA
- structural roles
- regulatory roles
- catalytic roles
What enzymes are involved in the process of capping?
5’-5’ linkage (reverse orientation - normally linked 5’ to 3’) at the 5’ end of all mRNAs
3 enzymes act in succession to remove the triphosphate of the original 5’ end
-phophatase (removes phosphates)
-guanyl transferase (adds guanosine)
-methyltransferase (adds methy groups)
What are the features of capping?
- Nascent pre-mRNA is capped after ~25-30 bases (RNA pol will pause)
- capping enzymes bind to the phosphorylated RNAPII CTD
How is the CTD involved in mRNA capping?
- unique feature that acts to recruit mRNA processing enzymes to the nascent transcript
- consists of heptad repeats, act as a platform to which other protein can bind
- phosphorylation of the CTD of RNA polymerase II recruits the processing enzymes for capping
What are the advantages of capping early?
RNA would be quickly degraded
What processing events occur at the 3’ end?
Polyadenylation
What are the features of the 3’ end of mRNA that make it different to the 5’ end?
- the 3’ end of mRNA is shorter than the original transcript, goes through a cleavage and addition of polyadenylation
- also differs as it ends up with a run of up to 200 adenosine residues
What are the reasons for adding a 5’ cap?
- Stability - most enzymes degrade a 5’ to 3’ linkage, but 5’ - 5’ linkage requires a special decapping enzyme
- stops transcript being degraded whilst being transcribed
- export from the nucleus - proteins in the nucleuss and cytoplasm specificially bind to the 5’ cap, mRNAs need to be moved to the cytoplasm unlike other RNAs
What are the features of the consensus sequence for cleavage and polyadenylation events?
AAUAAA at 5’ end-10-30 nucleotides-CA[cLEAVAGe]- end
- cleavage at the CA dinucleotide and addition of a -OH group
- Gu or U rich region is degraded in the nucleus
- Polyaddenylation (~250) occurs between CA dinucelotide and the -OH group
What are the proteins involved in cleavage and polyadenylation?
Cleavage stimulation factor F (CstF)
Cleavage and polyadenylation specificity factor (CPSF)
Poly-A-polymerase (PAP)
How are CstF, CPSF and other proteins (PABP, PAP) involved in cleavage and polyadenylation?
CstF and CPSF travel with the phosphorylated CTD tail of RNA polymerase II during trancription
- transfer to the 3’ end processing sequences on the RNA molecule once the sequences are transcribed
- interact with each other and form a loop bound to specific sites of DNA
- following cleavage PAP sequentially adds approximially 200 A nucleotides (using ATP as a precursor)
- poly-A-binding proteins (PABP) assemble onto the poly-A-tail
Why polyadentylate mRNA? Why not use some other method
Stability
-PA forms a circle with the CAP protein to protect the end of mRNA
Translatiion
-circularisation is important for the efficiency of translation
How is the importance of polyadenlyation reflected in some diseases?
- Thalassemias can be caused by mutations affecting the AAUAAA consescous sequence of α and β-globin gene
- Thrombophilla caused by mutations in the Prothrombin coagulation factor II (F2) gene. Causes raised plasma concentrations of pro-threombin due to increasses in the efficiency of polyadenylation
Describe how an increase in polyadenylation efficiency can cause disease (with example)
- Thrombophilla caused by mutations in the Prothrombin coagulation factor II (F2) gene
- Causes raised plasma concentrations of pro-threombin due to increasses in the efficiency of polyadenylation
- Normal F2 mRNA has unusual polyadenylation signals compared to efficiently processed mrNAs such as β-globin (HBB)
- Gain-of-function mutant scan increase the efficiency of polyadenylation meaning that more protein is present in the cell (raised plasma concentrations of pro-threombin)
What is the purpose of RNA splicing?
To remove introns from an mRNA primary transcript
