RNA base modifications and their regulatory roles 1

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