RNA modifications

What are RNA modifications?

Changes to the chemical composition of RNA molcules post-synthesis and can occur on all types of RNAs

What are the irreversible classes of RNA mods?

pseudouridylation (U to pseudo), editing (A → I; C → U)

What is m6A (methyl-6-adenosine)

The most abundant modification in mammalian mRNA, occurs at the N6 position of adenosine in RNA molecules

What enzymes are involved in m6A?

RNA methyl transferase (writer)

RNA demethylase (eraser)

RNA methyl binding protiens (reader)

What are the reversible classes of RNA modification

methylation (m6A)

How does MeRIP-seq detect m6A?

It fragments RNA, binds an antibody to target RNA, and does an immunoprecipitation to elude RNA bound to m6A. You then sequence it!

You then can further sequence it to determine where the m6A residue is based off a curve

m6A-seq for genome wide m6A detection

(I hate it here)

You take an RNA sample, immunosupress it and then generate cDNA from it.

More general RNA rules

Most m6A mRNAs have only one site, but some have 20 or more! Conserved between mouse and human.

Large exons are more likely to be modified

Genes that regulate development and cell fate specification are more likely to be modified in this method

Which sites are commonly m6A modified?

ACU

Functional outputs by different m6A readers

All this says is that different readers depending on their location change their functions? it literally gives us nothing to work with (slide wise)

They are also context dependent???

When are m6As added typically?

During translation and they are added co-transcriptionally with the help of histones

How do m6A help with large exons?

They help splicing, maybe?

how does m6A affect pri-miRNA regions?

It can inhibit the transition from pri to pre miRNAs. so stunts miRNA development

Pseudouridylation

Really abundant

Clevage of a glycosidic bond, rotation of the base, and base reattachment.

Catalyzed by pseudouridine synthases (PUS)

How does pseudouridine bind to other bases?

It is considered to be a universal base pairing partner

it can also form an extra hydrogen bond!

How does pseudouridine affect DNA sequencing?

It can be modified with CMC which can lead to the prevention of RNA into cDNA by reverse transcriptase

How many PUS proteins are there?

13, with 12 of them being RNA-independent

What can pseudouridylation affect RNA wise?

RNA structure, RNA binding protiens, tRNA and rRNA altering of decoding and amino acid incorporation and also altered tRNA selection by the ribosome

What the fuck is a snoRNA

small nucleolar RNAs

2 types: H/ACA box (pseudouridylation), C/D box snRNAs (2’ O methylation)

form snoRNPs by binding with snoRNA binding protiens

DKC1

Possible roles of pseudouridines

They can be involved in the splicesome and ribosome.

How do pseudouridines relate to vaccines?

pseudouridine prevents the acitvation of TLRs and prevent an interferon response, which is helpful to prevent the body from attacking mRNA vaccines (like COVID)

RNA editing

A post-transcriptional process tha modifies the primary RNA transcripts, thus creating new information in the RNA that is not encoded directly from the DNA

Yeah so this is just changing certian base pairs into other base pairs

A → I editing

Occurs on dsRNAs, inosine is typically recognized as guanosine

Mediated by ADAR! recognizies dsRNA substartes within repetitve elements like Alu sequences

GLUA2

So Q is changed into an R via the A → I editing method and prevents Ca+ from passing through the channel (good) unedited proteins dont do this (bad)

A → I editing on transcribed inverted repeats

Happens on Alu/SINE and LINE which lead to hairpins forming and ADAR to do its thing

How can A → eiditing regulate miRNA function

a: it doesnt

b: It prevents proper processing and leads to degredation in the nucleus

c: it prevents processing and suppresses Dicer step and doesnt form into mature RNA

d: it changes the target of the mRNA

endo-siRNA biogenesis and A → I biogenesis

Generally ADAR leads to less siRNA and even fewer functional copies

What are some of the things that a → I editing may induce?

Splicing and mRNA export

the mRNA export is via p54nrb

C → U editing

Only found in mammals, mediated by the AID/APOBEC family cytidine deaminases, and considered less frequent but more specific

How does c → U editing affect the editing of apolipoproteins?

mRNA of this gene is 14kb in human

Editing leads to a C → U change in exon 26 which creates a stop codon and production of a truncated form of the protein

Less than 1% ApoB transcripts are edited in liver, while over than 90% are edited in the intestine

Both long and short forms circulate in blood

What is the consensus sequence for c → u editing?

CAUCG? or CCAUCG?

How is c → U editing regulated?

By cell types and environmental factors