RNA base modifications and their regulatory roles 2

Question 2

Where do most M-6-A modifications occur?

Answer 2

They cluster at the start of the 3’ UTR

Question 3

What is the 3’UTR important for?

Answer 3

Regulating stability of mRNA, translation rate, location

Question 4

What do YTHDF1 and YTHDF2 do regarding M6A?

Answer 4

They read the M6A marks on the mRNAs

Question 5

Specific role of YTHDF1?

Answer 5

Speeds up translation rate of mRNAs that have M6A

Question 6

Specific role of YTHDF2?

Answer 6

Induces decay of mRNA that has M6A

Question 7

Where does YTHDF1 and YTHDF2 bind?

Answer 7

To M6A

Question 8

What is thought to be the role of M6A markers?

Answer 8

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

Question 9

Example of use of M6A?

Answer 9

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

Question 10

Why is 5MC harder to pinpoint than M6A?

Answer 10

It is less abundant

Question 11

What are the ways to find 5MC in mRNA?

Answer 11

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

Question 12

How does bisulfite sequencing work?

Answer 12

Treat DNA with bisulfite (converts unmethylated Cs to Us)
Sequence-> Us are read as Ts during PCR

Question 13

Issue with bisulfite treatment?

Answer 13

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

Question 14

What is M5C-RIP?

Answer 14

Similar to M6A-seq

Question 15

Issue with M5C-RIP and M6A-seq?

Answer 15

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

Question 16

How do catalytic crosslinking base methods work?

Answer 16

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 17

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

Answer 17

NSUN6

Question 18

Where does 5MC modificaitons cluster?

Answer 18

3’UTR

Question 19

What is inosine?

Answer 19

A base that forms via modifications of adenosine bases

Question 20

Why is inosine special?

Answer 20

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

Question 21

How is adenosine converted into inosine?

Answer 21

Enzymatically, by ADAR1 or ADAR2

Question 22

What is an inosine read as by translation machinery?

Answer 22

Guanosine

Question 23

What is an inosine read as by reverse transcriptase?

Answer 23

Guanosine

Question 24

How are inosine modifications found?

Answer 24

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

Question 25

Example of inosine modifications being useful?

Answer 25

In an AMPA receptor

Question 26

How is inosine important in the AMPA receptor?

Answer 26

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

Question 27

Effect of A-->I on AMPA receptor?

Answer 27

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 28

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

Answer 28

AMPA receptor wont close to hyperexcitability in the neuron will occur

Question 29

Condition caused by mutation in the ADAR2 receptor?

Answer 29

Epilepsy