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Gene regulation > RNA base modifications and their regulatory roles > Flashcards

RNA base modifications and their regulatory roles Flashcards

1
Q

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

A

Easier to purify and higher abundance of them

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2
Q

What has made it possible to detect mRNA modificaitons?

A

Next generation sequencing methosd

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3
Q

What is the transcriptome?

A

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

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4
Q

What is the most heavily modified RNA biotype?

A

tRNA

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5
Q

How frequently does tRNA modifications occur?

A

Every few bases

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6
Q

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

A

The anticodon

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7
Q

What is the role of the anticodon?

A

Recognise the mRNA

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8
Q

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

A

It can recognise more than one base entering the tRNA

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9
Q

Example of an anticodon recognition?

A

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

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10
Q

How to tRNAs recognise multiple different codon sequences?

A

Via modification of the wobble position of the anticodon

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11
Q

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

A

2

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12
Q

What is a formyl group?

A

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

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13
Q

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

A

The cytosine

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14
Q

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

A

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

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15
Q

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

A

Cytosine is converted to 5-methylcytosine

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16
Q

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

A

NSUN3

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17
Q

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

A

5-methylcytosine is converted to 5-formylcytosine

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18
Q

What enzyme catalyses the formation of 5-formylcytosine?

A

ABH1

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19
Q

What caused the mitochondrial disease?

A

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

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20
Q

What is the epitranscriptome?

A

Modifications that are being done on mRNA molcules

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21
Q

What is the most prevalent mRNA modification?

A

methyl-6-adenosine

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22
Q

Second most prevalent mRNA modification?

A

Methyl-5-cytosine

23
Q

How is the transcriptome analysed using NGS?

A

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

24
Q

Why is normal transcriptome analysis problematic when analysing RNA modifications?

A

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

25
Q

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

A

M-6-A-seq

26
Q

Steps of M-6-A-seq?

A

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

27
Q

Where do most M-6-A modifications occur?

A

They cluster at the start of the 3’ UTR

28
Q

What is the 3’UTR important for?

A

Regulating stability of mRNA, translation rate, location

29
Q

What do YTHDF1 and YTHDF2 do regarding M6A?

A

They read the M6A marks on the mRNAs

30
Q

Specific role of YTHDF1?

A

Speeds up translation rate of mRNAs that have M6A

31
Q

Specific role of YTHDF2?

A

Induces decay of mRNA that has M6A

32
Q

Where does YTHDF1 and YTHDF2 bind?

A

To M6A

33
Q

What is thought to be the role of M6A markers?

A

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

34
Q

Example of use of M6A?

A

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

35
Q

Why is 5MC harder to pinpoint than M6A?

A

It is less abundant

36
Q

What are the ways to find 5MC in mRNA?

A

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

37
Q

How does bisulfite sequencing work?

A
38
Q

Issue with bisulfite treatment?

A

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

39
Q

What is M5C-RIP?

A

Similar to M6A-seq

40
Q

Issue with M5C-RIP and M6A-seq?

A

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

41
Q

How do catalytic crosslinking base methods work?

A

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

42
Q

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

A

NSUN6

43
Q

Where does 5MC modificaitons cluster?

A

3’UTR

44
Q

What is inosine?

A

A base that forms via modifications of adenosine bases

45
Q

Why is inosine special?

A

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

46
Q

How is adenosine converted into inosine?

A

Enzymatically, by ADAR1 or ADAR2

47
Q

What is an inosine read as by translation machinery?

A

Guanosine

48
Q

What is an inosine read as by reverse transcriptase?

A

Guanosine

49
Q

How are inosine modifications found?

A

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

50
Q

Example of inosine modifications being useful?

A

In an AMPA receptor

51
Q

How is inosine important in the AMPA receptor?

A

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

52
Q

Effect of A–>I on AMPA receptor?

A

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

53
Q

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

A

AMPA receptor wont close to hyperexcitability in the neuron will occur

54
Q

Condition caused by mutation in the ADAR2 receptor?

A

Epilepsy

Gene regulation (12 decks)

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