L8 - Post-transcriptional control of gene expression II Flashcards
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What is RNA editing/modification?
RNA editing – nucleotide alterations which result in different or additional nucleotides in the mature RNA
Changes the coding sequence of mRNAs
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Where does editing occur?
Occurs in all 3 major classes of RNA – mRNA, tRNA & rRNA
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What are the 2 classes of editing?
Insertion/deletion – can be a few nucleotides or a whole line
Modification (eg. A to I, C to U, U to C)
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Marked nucleotides
Adenosine can be methylated at the 6 position to give m6A – it is reversible
This changes the properties of the base, and it marks the RNA
– e.g. should be translated or degraded or moved to a different place etc.
Adding methyl group to base changes the base and changes the property of the RNA
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Altered activity
Uracil can be converted to pseudo-uridine – the same base, but it has been turned around and put back on, so instead of CN bond have a CC bond
Change the bonding in the base changes the properties by turning it around and end up with pseudo-uridine
One of the most common modifications in RNA
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How does the change from uracil to pseudo-uridine cause altered activity?
Pseudo-uridine can base pair with adenosine, but instead of a C in 5 position we have a NH which has the potential to do extra bonding, so can stabilise base pairing
So we have altered activity
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Altered identity
Adenosine, can be converted by deamination to inosine, which is read as G, so looks like a G instead of an A
We can alter the identity of a base
Can remove amino groups, replacing it with =O which the means the A is I, I is read as G and this pairs with C
Same with C which changes to U, which results in an AU base pair
If this is in the middle of a codon it changes the sequence which could be very dramatic
Can introduce stop codons, and or change the identity of the amino acid being coded
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Effects of mRNA editing
The markers will change some of the properties of RNA, will change where it localised, how stable it is etc
But if you change the base identity, can start really changing the RNA
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What is N6-methyladenosine?
Most prevalent internal modification in eukaryotic mRNA
Adenosine converted to m6A by the addition of a methyl group
Regulated by writers, readers, & erasers
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How does N6-methyladenosine affect function?
Base pairing can still occur – that property is not changed
m6A is recognised by regulatory factors & readers that can change the properties of the RNA
Can allow protein binding or can stop proteins binding
Can affect things like stability & degradation
Very context specific – depends on where the RNA is & what it does
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RNA editing by deamination
Base conversion by deamination
Adenosine or cytosine, by enzymatic deamination, get the amine group removed at the top, ending up with inosine or uracil
Inosine recognised as guanosine – not identical – equivalent of an A to G change
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How does deamination change adenosine?
Ends up with inosine
Inosine recognised as guanosine – not identical – equivalent of an A to G change
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How does deamination change cytosine?
Ends up with uracil
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ApoB mRNA editing
Editing carried out by the APOBEC-1 enzyme (linked to cholesterol control, cancer development and inhibition of viral replication)
2 types of ApoB
• ApoB-48
• ApoB-100
Both forms circulate in the blood but have different functions
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How are ApoB-48 & ApoB-100 different?
ApoB-100 - from no editing in the liver
• Creates a 2 domain protein
ApoB-48 - from editing in the intestine
• C converted to U so we get a stop codon before the actual stop codon
• Leads to a truncated protein
• ApoB-48 protein produced that is lacking the LDL-receptor binding protein
By 1 little change we have produced 2 different proteins
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A to I editing in the Q/R site of glutamate receptors
Lots of neuronal proteins have A to I editing
Editing yields decrease in Ca2+ permeability of channels containing the ‘R’ version
Q version is encoded in the genome, but we can end up with the R version
R version allows sodium through but doesn’t let the cell take up calcium – this affects neurotransmission
Extent of editing varies in different parts of the body and the brain
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What do glutamate receptors do?
Important in the influx of sodium & calcium into the neurone
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What is A to I editing in the Q/R site of glutamate receptors carried out by?
Editing carried out by ADAR2 (adenosine deaminase acting on RNA)
Mutations in the mouse ADAR2 gene lead to seizures, post-natal death, neurodegeneration in the hippocampus
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RNA modification - ribose modification
Affects the properites of mRNA
When capping takes place we also get methylation of the first 2 nucleotides at the 2’ position
– 2’-O-methylation
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Modification of rRNA
2 subunits joined together, & the enzymatic activitiy takes place between the 2 subunits
mRNA threads through the A, P & E sites – where the tRNAs are sitting
RNA contains a lot of modifications – modifications are clustered around the active sites
Export of RNA from the nucleus
mRNA once produced needs to enter the cytoplasm where it can be translated
Not a simple case of diffusion
Got to get mRNA through the pore
mRNA is usually localised to specific regions of cells & translated in specific regions which can be fundamental for cell development
RNA EXPORT
The nuclear pore
The inner pore is very hydrophilic
RNA is a nucleic acid so it hydrophilic
So not easy to get through the nuclear pore
Regulated process – need adaptor proteins & factors which are going to bring the molecule in & dock with the basket structure on the nuclear side
RNA EXPORT
What are export factors?
All RNAs are processed, matured & assembled with export factors
Export of things through the pore is protein mediated
Often different factors are needed for each class of RNA
Assemble complexes of RNA and protein to allow the RNA to be exported out of the nucleus, through the nuclear pore into the cytoplasm
RNA EXPORT
What are the groups of export factors?
Exportins - most important ones
Adaptor proteins
GTPases
