L8 - Post-transcriptional control of gene expression II

Question 1

EDITING

What is RNA editing/modification?

Answer 1

RNA editing – nucleotide alterations which result in different or additional nucleotides in the mature RNA

Changes the coding sequence of mRNAs

Question 2

EDITING

Where does editing occur?

Answer 2

Occurs in all 3 major classes of RNA – mRNA, tRNA & rRNA

Question 3

EDITING

What are the 2 classes of editing?

Answer 3

Insertion/deletion – can be a few nucleotides or a whole line

Modification (eg. A to I, C to U, U to C)

Question 4

EDITING

Marked nucleotides

Answer 4

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

Question 5

EDITING

Altered activity

Answer 5

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

Question 6

EDITING

How does the change from uracil to pseudo-uridine cause altered activity?

Answer 6

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

Question 7

EDITING

Altered identity

Answer 7

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

Question 8

EDITING

Effects of mRNA editing

Answer 8

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

Question 9

EDITING

What is N6-methyladenosine?

Answer 9

Most prevalent internal modification in eukaryotic mRNA

Adenosine converted to m6A by the addition of a methyl group

Regulated by writers, readers, & erasers

Question 10

EDITING

How does N6-methyladenosine affect function?

Answer 10

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

Question 11

EDITING

RNA editing by deamination

Answer 11

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

Question 12

EDITING

How does deamination change adenosine?

Answer 12

Ends up with inosine

Inosine recognised as guanosine – not identical – equivalent of an A to G change

Question 13

EDITING

How does deamination change cytosine?

Answer 13

Ends up with uracil

Question 14

EDITING

ApoB mRNA editing

Answer 14

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

Question 15

EDITING

How are ApoB-48 & ApoB-100 different?

Answer 15

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

Question 16

EDITING

A to I editing in the Q/R site of glutamate receptors

Answer 16

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

Question 17

EDITIING

What do glutamate receptors do?

Answer 17

Important in the influx of sodium & calcium into the neurone

Question 18

EDITING

What is A to I editing in the Q/R site of glutamate receptors carried out by?

Answer 18

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

Question 19

EDITING

RNA modification - ribose modification

Answer 19

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

Question 20

EDITING

Modification of rRNA

Answer 20

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

Question 21

Export of RNA from the nucleus

Answer 21

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

Question 22

RNA EXPORT

The nuclear pore

Answer 22

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

Question 23

RNA EXPORT

What are export factors?

Answer 23

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

Question 24

RNA EXPORT

What are the groups of export factors?

Answer 24

Exportins - most important ones

Adaptor proteins

GTPases

Question 25

What happens once mRNA is in the cytoplasm?

Answer 25

It needs to be localised

Question 26

Why do we need to localise mRNA?

Answer 26

Localised protein synthesis – can control where the protein is produced, producing in wrong place be toxic or wasteful

Generate cell polarity

Prevents expression in the wrong place

Promotes efficiency of subsequent protein targeting

Local control of translation

Question 27

Localisation of mRNAs during drosophila development

Answer 27

Grk by nucleus
Osk just at the cap
Bcd in the central part

This is so the cell develops with the right anterior-posterior & D-V polarity

Question 28

Localisation of dendritic mRNAs

Answer 28

mRNA is travelling along the dendritic spine

Go from the nucleus & localises to the synapse where it will be locally translated

RNA is blocked from being translated until it gets to the right destination

Question 29

Diffusion-based localisation

Answer 29

Local entrapment – diffusion-based method

mRNA is produced & comes out into the cytoplasm, & is enriched in the area as it becomes trapped there

Question 30

Active transport-based localisation

Answer 30

mRNA actively moves through the cell using the cytoskeleton

Is transported with a transport factor

Factor stays bound to the mRNA - held in a state where it can’t be translated

mRNA binds onto cytoskeletal factors, binds and then is translated along the cytoskeleton

Active process, anchored and held at defined site for translation

Can either get to anchorage site by passive diffusion or active transport