RNA base modifications and their regulatory roles

Question 1

Why were tRNAs and rRNAs the main types of RNA studied early on?

Answer 1

Easier to purify and higher abundance of them

Question 2

What has made it possible to detect mRNA modificaitons?

Answer 2

Next generation sequencing methosd

Question 3

What is the transcriptome?

Answer 3

All the mRNA that can be transcribed by a cell or organism

Question 4

What is the most heavily modified RNA biotype?

Answer 4

tRNA

Question 5

How frequently does tRNA modifications occur?

Answer 5

Every few bases

Question 6

Which part of the tRNA is the wobble position located on?

Answer 6

The anticodon

Question 7

What is the role of the anticodon?

Answer 7

Recognise the mRNA

Question 8

What does the wobble position do compared to the other two positions?

Answer 8

It can recognise more than one base entering the tRNA

Question 9

Example of an anticodon recognition?

Answer 9

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

Question 10

How to tRNAs recognise multiple different codon sequences?

Answer 10

Via modification of the wobble position of the anticodon

Question 11

How many codons does human mitochondrial tRNA need to be able to recognise?

Answer 11

2

Question 12

What is a formyl group?

Answer 12

a carbon double bonded to an oxygen, single bonded to a H and single bonded to an R group

Question 13

Which part of the methionine anticodon (CAU) is modified so it can interact with AUA?

Answer 13

The cytosine

Question 14

How is the cytosine in CAU modified so it can interact with the A in AUA?

Answer 14

It has a formyl group added onto it at the 5th position, forming 5-formylcytosine

Question 15

What is the first step in forming 5-formylcytosine from cytosine?

Answer 15

Cytosine is converted to 5-methylcytosine

Question 16

What enzyme catalyses the formation of 5-methylcytosine from cytosine?

Answer 16

NSUN3

Question 17

What happens after 5-methylcytosine is formed in the formation of 5-formylcytosine?

Answer 17

5-methylcytosine is converted to 5-formylcytosine

Question 18

What enzyme catalyses the formation of 5-formylcytosine?

Answer 18

ABH1

Question 19

What caused the mitochondrial disease?

Answer 19

NSUN3 enzyme is mutated–> no formation of 5-methylcytosine–>no 5-formylcytosine–> codon not recognised–> no formation of protein

Question 20

What is the epitranscriptome?

Answer 20

Modifications that are being done on mRNA molcules

Question 21

What is the most prevalent mRNA modification?

Answer 21

methyl-6-adenosine

Question 22

Second most prevalent mRNA modification?

Answer 22

Methyl-5-cytosine

Question 23

How is the transcriptome analysed using NGS?

Answer 23

mRNA is isolated–> converted into DNA using RT–> amplified with PCR–> illumina sequencing

Question 24

Why is normal transcriptome analysis problematic when analysing RNA modifications?

Answer 24

A modification on the RNA is not copied into DNA by reverse transcriptase

Question 25

What type of sequencing is used to look for methyl-6-adenosine?

Answer 25

M-6-A-seq

Question 26

Steps of M-6-A-seq?

Answer 26

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

Question 27

Where do most M-6-A modifications occur?

Answer 27

They cluster at the start of the 3' UTR

Question 28

What is the 3'UTR important for?

Answer 28

Regulating stability of mRNA, translation rate, location

Question 29

What do YTHDF1 and YTHDF2 do regarding M6A?

Answer 29

They read the M6A marks on the mRNAs

Question 30

Specific role of YTHDF1?

Answer 30

Speeds up translation rate of mRNAs that have M6A

Question 31

Specific role of YTHDF2?

Answer 31

Induces decay of mRNA that has M6A

Question 32

Where does YTHDF1 and YTHDF2 bind?

Answer 32

To M6A

Question 33

What is thought to be the role of M6A markers?

Answer 33

To generate a high burst of translation of specific mRNA sequences but only for a short time

Question 34

Example of use of M6A?

Answer 34

Synaptic signalling--> proteins need to be translated within synaptic locations in response to activation--> needs to be acute

Question 35

Why is 5MC harder to pinpoint than M6A?

Answer 35

It is less abundant

Question 36

What are the ways to find 5MC in mRNA?

Answer 36

Bisulfite sequencing, M5C-RIP, Aza-IP, miCLP

Question 37

How does bisulfite sequencing work?

Question 38

Issue with bisulfite treatment?

Answer 38

It causes degradation of the RNA as RNA is less stable than DNA, and you can get a lot ae +ves

Question 39

What is M5C-RIP?

Answer 39

Similar to M6A-seq

Question 40

Issue with M5C-RIP and M6A-seq?

Answer 40

Dont know where the modification is, only that it is somewhere in the 100 base fragment

Question 41

How do catalytic crosslinking base methods work?

Answer 41

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

Question 42

Which enzyme can methylate a particular consensus sequence found in 3' UTRs?

Answer 42

NSUN6

Question 43

Where does 5MC modificaitons cluster?

Answer 43

3'UTR

Question 44

What is inosine?

Answer 44

A base that forms via modifications of adenosine bases

Question 45

Why is inosine special?

Answer 45

It results in an edit in the coding sequence that can change the AAs present in the translated protein

Question 46

How is adenosine converted into inosine?

Answer 46

Enzymatically, by ADAR1 or ADAR2

Question 47

What is an inosine read as by translation machinery?

Answer 47

Guanosine

Question 48

What is an inosine read as by reverse transcriptase?

Answer 48

Guanosine

Question 49

How are inosine modifications found?

Answer 49

As reverse transcriptase reads inosine as guanosine, just look for guanosines that have come from an adenosine

Question 50

Example of inosine modifications being useful?

Answer 50

In an AMPA receptor

Question 51

How is inosine important in the AMPA receptor?

Answer 51

A subunit of the receptor is a target of A-->I editing. Results in an arginine being encoded for instead of a glutamine

Question 52

Effect of A-->I on AMPA receptor?

Answer 52

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

Question 53

What happens if there is a mutation in the ADAR2 enzyme?

Answer 53

AMPA receptor wont close to hyperexcitability in the neuron will occur

Question 54

Condition caused by mutation in the ADAR2 receptor?

Answer 54

Epilepsy