RNA modifications

How many RNA modification are there?

~100, more than DNA, chemically diverse

Where do modifications occur?

On tRNAs and rRNAs, which are abundant in cells so easy to detect. Also detected in mRNAs using next generational sequencing.

Most heavily modified RNA molecules

tRNAs

How often do tRNA modifications occur?

Every few nucleotides, more chemically diverse than DNA modifications

Most heavily modified site

position 34 of tRNAs - wobble position

What is the wobble position?

3rd position of anticodon site in tRNA

Recognise more than 1 codon in mRNA.

The G recognises both C and U by nucleotide modifications

Allow non-standard Watson-Crick base pairing

example in human mtRNA

mitochondrial disease due to mutation in enzyme that catalyses the modification. Mutation - not catalysed.

Normal wobble modification in methionine (human mitochondrial tRNA)

cytosine → 5-formyl cytosine

Important bc they encode methionine so initiators for translation

CAU recognises AUA and AUG

Enzyme for cytosine → 5-methylcytosine

(In methionine mt-tRNA C34)

NSUN3

Enzyme for 5-methylcytosine → 5-formylcytosine

ABH1

What happens if NSUN3 not present/mutated?

5-methylcytosine and 5-formylcytosine no longer form

What happens when 5-formylcyotosine not present?

Initiation of translation is inhibited

Leads to mitochondrial dysfunction

What is the epitranscriptome?

chemical modifications on RNA

Most prevalent modification type in vertebrate epitranscriptome

methyl-6-Adenosine then 5mC and inosine

How are they detected?

NGS

Process of transcriptome sequencing

extract + purify mRNA → convert to DNA using reverse transcriptase → PCR amplify → sequence

Problem with process of transcriptome sequencing

RNA modifications lost when converted to DNA

Method used to map 6-methyladenosine

m6A-Seq

How does m6A-Seq work?

fragmentation - manipulated so they are a certain size (100nt) → purify m6A fragments with immunoprecipitation with anti m6a antibody → sequencing → gives 100nt windows of there m6a occurs.

Where do m6a marks cluster?

start of 3’UTRs, after the stop codon

Which proteins bind to m6a and what do they do?

YTHDF1 → elevates translation

YTHDF2 → decay of mRNA

What does this mean?

When m6a is present, get a short burst of translations

Why is this important?

e.g. synapses need short bursts of signalling

Why is mapping of 5mc challenging?

Not as abundant as m6a

Why is bisulphite sequencing inaccurate in RNA?

You get incomplete conversions → false positives

Methods to map 5mc

1. bisulphite sequencing

2. similar to m6a-seq

3. Aza-IP

4. methylation iCLIP

3 and 4 are cross linking methods

How do catalytic cross linking methods work?

induce a cross-link between the enzyme catalysing the modification

incubate mRNA with 5-Aza-C → overexpresses methyltransferase → enzyme methylated → becomes cross-linked → sequencing detects cross-link

How does miCLIP work?

modify the enzyme → get a cross link when it methylates the cytosine → customise sequencing to map where methylation occurs.

Cross-link allows you to map to nucleotide resolution

Where does 5mc occur?

In the 3’UTR, probably has regulatory roles

How do inosine bases form?

via enzymatic modification of adenosine bases

Why is inosine special?

results in an edit in the coding sequence that can change the amino acids present in the translated protein

Which enzymes convert A to I

ADAR1 or ADAR2

What happens when AUA → AUI

I recognised as G, changes the amino acid.

How is inosine detected?

Reverse transcriptases also read inosines as guanosines → there will be differences compared to the exprected sequence.

A→G substitution indicates an A→I conversion. rare in vertebrantes.

Why is inosine important in AMPA receptors?

inosine edit causes glutamine→arginine

Arginine sits in the channel to close it.

Regulates opening + closing of channel.

What happens without the edit in AMPA?

channel permanently open → hyperexcitability → epilepsy