RNA base modifications and their regulatory roles Flashcards
Why were tRNAs and rRNAs the main types of RNA studied early on?
Easier to purify and higher abundance of them
What has made it possible to detect mRNA modificaitons?
Next generation sequencing methosd
What is the transcriptome?
All the mRNA that can be transcribed by a cell or organism
What is the most heavily modified RNA biotype?
tRNA
How frequently does tRNA modifications occur?
Every few bases
Which part of the tRNA is the wobble position located on?
The anticodon
What is the role of the anticodon?
Recognise the mRNA
What does the wobble position do compared to the other two positions?
It can recognise more than one base entering the tRNA
Example of an anticodon recognition?
A G is in the anticodon and it needs to be able to recognise and form an interaction with both a C and a U
How to tRNAs recognise multiple different codon sequences?
Via modification of the wobble position of the anticodon
How many codons does human mitochondrial tRNA need to be able to recognise?
2
What is a formyl group?
a carbon double bonded to an oxygen, single bonded to a H and single bonded to an R group
Which part of the methionine anticodon (CAU) is modified so it can interact with AUA?
The cytosine
How is the cytosine in CAU modified so it can interact with the A in AUA?
It has a formyl group added onto it at the 5th position, forming 5-formylcytosine
What is the first step in forming 5-formylcytosine from cytosine?
Cytosine is converted to 5-methylcytosine
What enzyme catalyses the formation of 5-methylcytosine from cytosine?
NSUN3
What happens after 5-methylcytosine is formed in the formation of 5-formylcytosine?
5-methylcytosine is converted to 5-formylcytosine
What enzyme catalyses the formation of 5-formylcytosine?
ABH1
What caused the mitochondrial disease?
NSUN3 enzyme is mutated–> no formation of 5-methylcytosine–>no 5-formylcytosine–> codon not recognised–> no formation of protein
What is the epitranscriptome?
Modifications that are being done on mRNA molcules
What is the most prevalent mRNA modification?
methyl-6-adenosine
Second most prevalent mRNA modification?
Methyl-5-cytosine
How is the transcriptome analysed using NGS?
mRNA is isolated–> converted into DNA using RT–> amplified with PCR–> illumina sequencing
Why is normal transcriptome analysis problematic when analysing RNA modifications?
A modification on the RNA is not copied into DNA by reverse transcriptase
What type of sequencing is used to look for methyl-6-adenosine?
M-6-A-seq
Steps of M-6-A-seq?
Collect mRNA–> Fragment it into 100 nucleotide fragments–> use a tagged antibody against M-6-A–> isolate positive fragments–> PCR and illumina sequence them. All those fragments would have had the M6A
Where do most M-6-A modifications occur?
They cluster at the start of the 3’ UTR
What is the 3’UTR important for?
Regulating stability of mRNA, translation rate, location
What do YTHDF1 and YTHDF2 do regarding M6A?
They read the M6A marks on the mRNAs
Specific role of YTHDF1?
Speeds up translation rate of mRNAs that have M6A
Specific role of YTHDF2?
Induces decay of mRNA that has M6A
Where does YTHDF1 and YTHDF2 bind?
To M6A
What is thought to be the role of M6A markers?
To generate a high burst of translation of specific mRNA sequences but only for a short time
Example of use of M6A?
Synaptic signalling–> proteins need to be translated within synaptic locations in response to activation–> needs to be acute
Why is 5MC harder to pinpoint than M6A?
It is less abundant
What are the ways to find 5MC in mRNA?
Bisulfite sequencing, M5C-RIP, Aza-IP, miCLP
How does bisulfite sequencing work?
Issue with bisulfite treatment?
It causes degradation of the RNA as RNA is less stable than DNA, and you can get a lot ae +ves
What is M5C-RIP?
Similar to M6A-seq
Issue with M5C-RIP and M6A-seq?
Dont know where the modification is, only that it is somewhere in the 100 base fragment
How do catalytic crosslinking base methods work?
Incubate the mRNA with a chemical that tags the nucleotide–> when the enzyme comes to methylate it it will form a permanent cross link–> can determine exactly what nucleotide is being modified
Which enzyme can methylate a particular consensus sequence found in 3’ UTRs?
NSUN6
Where does 5MC modificaitons cluster?
3’UTR
What is inosine?
A base that forms via modifications of adenosine bases
Why is inosine special?
It results in an edit in the coding sequence that can change the AAs present in the translated protein
How is adenosine converted into inosine?
Enzymatically, by ADAR1 or ADAR2
What is an inosine read as by translation machinery?
Guanosine
What is an inosine read as by reverse transcriptase?
Guanosine
How are inosine modifications found?
As reverse transcriptase reads inosine as guanosine, just look for guanosines that have come from an adenosine
Example of inosine modifications being useful?
In an AMPA receptor
How is inosine important in the AMPA receptor?
A subunit of the receptor is a target of A–>I editing. Results in an arginine being encoded for instead of a glutamine
Effect of A–>I on AMPA receptor?
Normally, in the unedited state, it remains open so Ca2+ can flow through. With the edit it results in a closed conformation–> can change the open or closed state of the receptor
What happens if there is a mutation in the ADAR2 enzyme?
AMPA receptor wont close to hyperexcitability in the neuron will occur
Condition caused by mutation in the ADAR2 receptor?
Epilepsy
