Lecture 6

Question 1

RNA bases also contain

Answer 1

Covalent modifications

Question 2

Which have more covalent modifications: DNA or RNA

Answer 2

RNA

Question 3

Historically RNA modifications associated with which type ?

Answer 3

• tRNA

- rRNA

Question 4

What is used to detect these modifications made to RNA?

Answer 4

next generation sequencing

Question 5

Epitranscriptonomics?

Answer 5

RNA expressed within a transcriptome

Question 6

Transcriptome ?

Answer 6

everything expressed with a cell

Question 7

most heavily modified RNA?

Answer 7

tRNA’s

Question 8

Position 34 importance with tRNA

Answer 8

WOBBLE POSITION:1st position within the anticodon in which multiple modifications take place.

Question 9

Modifications require?

Answer 9

ENERGY

Question 10

Wobble position modification

Answer 10

non waston-crick base paring at the 1st position

Question 11

Mitochondrial disease caused by?

Answer 11

NSUM3 gene not methylated leading to a mutagenised gene

Question 12

Do mitochondria express their own tRNA?

Answer 12

yes

Question 13

What modification is required in a methionine, tRNA complex?

Answer 13

F5c for AUA AUG

tRNA recognises both and forms a stable interaction

Question 14

NSUM3 function

Answer 14

methylation at the 5th position

Question 15

ABH1 function

Answer 15

5formylcytosone formation

Question 16

Experiment involving NSUM3 mutation?

Answer 16

• extract skin cells containing this NSUM3 mutation, purify (so you only have the mitochondria)
• Extract the protein
• Load onto a gel
• Compare to the wild type and can see that less NSUM3 expressed

Question 17

Stable transfection of NSUM3

Answer 17

lead to restored function in the mitochondria

Question 18

Most common mRNA modification

Answer 18

m6a

Question 19

Method for detection of m6a modification

Answer 19

Isolate mRNA
convert into cDNA using reverse transcriptase
PCR amplify
NGS (illumina)

Question 20

m6A sequencing method ?

Answer 20

• Antibody raised against m6A
• Antibodys bind
• Isolate them
• purify
• Wash away unmodified antibodies
• Reference to genome

Question 21

YTHDF2 function?

Answer 21

Decay, regulates translation

Question 22

Rate of translation which occurs in RNA with the modification m6A

Answer 22

Short Lived, quick production

Question 23

Localsied where? m6A ?

Answer 23

Neurones

Question 24

5MC is less abundant where? Therefore?

Answer 24

In mRNA

a more sensitive deception method required

Question 25

Why don’t we use bisulphate to detect the presence of 5mc?

Answer 25

usually creates a false positive (lots of normal cytosines don’t convert)

Question 26

Why don’t we use fragmentation method for the section of 5mc either?

Answer 26

Fragmentation can occur anywhere within the fragment (large fragments, we are unsure on the location)

Question 27

Solution to detect the presence of 5mc in mRNA?

Answer 27

express an enzyme which catalysis reaction

Catalytic crosslink- freeze when an enzyme substrate complex, purify and locate single location in which it occurs (NT)

Question 28

nucleotide sequence in which 5mc modification occurs

Answer 28

ctcca

Question 29

RNA editing method?

Answer 29

1) inosine bases form via enzymatic modifications to adenosine bases
2) Translation occurs which forms a G base.
3) Compare to original A instead of G= been editied

Question 30

ADAR1 AND ADAR2 function?

Answer 30

A to I BASE

Question 31

What else reads inosine as guanines ? What does this mean

Answer 31

Reverse transcriptases

can compare to Normal DNA and see the differences in which There are g’s instead of A’s

Question 32

Where is A-I editing rare?

Answer 32

Mamallian

Question 33

Example of where A-1 editing does occur in mammals

Answer 33

AMPA RECEPTORS

• ca2+ channel with glutamate and glycine binding sites
• An influx on Ca2+ ions causes excitation of the synapse
• Adenosine to Arg edit causes the channel to close.
• Without this = neurological function (e.g epilepsy)
• This allosteric inhibition of the ca2+ channel enables the correct excitation of the channels