Lecture 5 - Pre-mRNA And ncRNA Processing Flashcards
What is on the 5’ end of every mRNA?
A cap
Why is the CTD tail important for RNA pol II?
scaffold for RNA processing proteins, regulates phosphorylation and is essential for life
The CTD tail recruits capping enzymes - how does this happen after the nucleotides are synthesised? What happens to the cap?
5’ triphosphate of the primary transcript is cleaved and a guanosine residue is added via 5’-5’ linkage. The guanosine cap is methylated
What happens if the mRNA is not capped?
The mRNA is degraded
What are the functions of the 5’ cap to mRNA?
Protects from degradation, promotes pre-mRNA splicing, needed for export from the nucleus and required for efficient translation in the cytoplasm
What produces RNA’s which do not have a cap?
Pol I and pol III
Polydadenlation and cleavage factors are also recruited to the CDT tail of pol II. What are the 2 important factors recruited here?
CPSF - cleavage and polyadenylation specificity factor
CstF - cleavage stimulating factor
When is CPSF and cstF recruited to the tail?
After phosphorylation of certain amino acids
What happens when the mRNA with the correct signal for termination goes past the CTD tail?
The CPSF and cstF will bind
What is the termination signal in the mRNA?
AAUAAA
What happens to the RNA after CPSF and cstF have bound?
It cleaves and recruits a polyA binding protein and a non-templates tail.
What does the recruitment of poly-A polymerase (PAP) and the template do to the mRNA?
Stabilises it
Is the poly-A tail encoded into the RNA?
No
You have cleaved the RNA tail and added a polyA tail on for protection but how does the ribosome know to stop translating?
Could be that the ribosome does not have a cap and is therefore being degraded
What is splicing?
The exons get transcribed but the introns need to be removed and spliced out
What do you need to splice?
enzymes for cutting and then join the two ends together
what type of process is splicing?
Catalytic
what does splicing form?
An intron lariat -
How does splicing then happen?
An A residue in the intron nucleophilic attacks the 5’ splice site creating a lariat structure (a loop) then the 5’ exon is joined to the 3’ exon
How is the 5’ and 3’ splice sites recognised?
Specific sequences both minor and major
How do proteins recognise specific sequences in RNA?
Through contacts between amino acids and bases or stacking interactions.
Or through a protein using a guide RNA (a ribonucleoprotein complex)
How does the proteins identify the sequence in splicing?
protein using a guide RNA (a ribonucleoprotein complex)
What is the names of the ribonucleoproteins involved in splicing?
U1, U2, U3, U4, U5 and U6.
The ribonucleoproteins involved in splicing are called what?
SnRNPs - small nuclear ribonucleic proteins
What do snRNPs recognise?
5’ splice site and branch site.
What do the snRNPs help do?
They help catalyse RNA cleavage and joining reactions
What does U1 recognise?
5’ splice site
What does BBP and U2Af bind to?
Branch point and 3’ splice site - starts making loop
What does U2 do?
Displaces BBP, binds to branch point making the A bulge
What does U4, U5, and U6 do?
They join the complex and U6 displaces U1 -
What does U4 specifically do?
Is displaced causing active site formation and catalysis
What does U5 specifically do?
Brings 2 exons together = final reaction
What do U6 and U2 bridge?
The 5’ and 3’
Can introns self cleave in some cases?
Yes
Where is nuclear free pre-mRNA splicing derived from?
The self cleave mechanism
Alternative splicing - can this be constitutive?
Yes -
What is alternative splicing?
The idea that you can make functional mature messanger RNA from a locus due to the combination of exons put together
Can alternative splicing be used as a regulatory mechanisms.
yes
Why is alternative splicing important?
Evolution of new proteins
How do we define which exons should be joined together?
Regulatory proteins
What is one type of regulatory proteins?
SR proteins
What do SR proteins do?
They bind to exons and contact splicing machinery (exonic splicing enhancers - ESEs). This helps cooradinate splicing in specific areas
What is the regulatory protein which binds to RNA but not the splicing machinery?
hnRNPs (heterogenous ribonucleoproteins) these are intronic/ exonic splicer silencer
What do SR proteins do?
Promotes splicing
What do hnRNPs do?
Inhibits splicing
Can splicing be regulated in a cell specific way?
yes - if there is a repressor near a splice site if a repressor is there then no splicing happens but if a repressor is not there splicing happens.
This is the same with activators
Can alternative splicing affect the untranslated regions as well as reading frames?
yes
Why is splicing medically relevant?
Many point mutation from splicing cause inherited human diseases
How does mutations impact alternative splicing?
- an exon migt get skipped
- the normal splice site is altered and another cryptic splice site is used.
- a mutation could just cause a new splice site to be used.
What happens when a mutation causes an exon to get skipped?
It will just join with the third exon
What is a cryptic splice site?
Sequence that has the potential for interacting with the spliceosome
Example of a splicing defect - deficiency’s in STAT-1. What was wrong with the splice site?
Mutation in the donor splice site skipping exon 3 - this makes the gene smaller and so moves through the gel quicker.
MRNA isn’t the only product of Pol II what are others?
MicroRNA’s
What are microRNA’s?
a class of non-coding RNA that are produced in a pol II transcripts (normally in the introns) before being moved into the cytoplasm.
How to microRNA’s moved to the cytoplasm? First stop the nucleus
Form a stem loop structure which is recognised by enzymes in the micro-processor of the nucleus (DROSHA and DGCR8)
What happens once the microRNA has been processed by DROSHA and DGCR8?
They are cleaved making a pre-miRNA which can go to the cytoplasm
What happens to pri-miRNA in the cytoplasm?
Processed by DICER1 to make it smaller and it then binds to AGO1-4
What happens to pri-miRNA once it is in the AGO1-4?
One strand is kept in the miRISC
Where does the miRISC take the single stranded miRNA?
To the mRNA to regulate it
What happens to the mRNA in the miRISC
The miRNA is a guide and can inhibit translation and mRNA degradation processes
Where can microRNA’s be produced from?
Exons and introns
