Lecture 6 - Regulatory Mechanisms For Quality Control And Turnover Of Pre-mRNA

Question 1

Why do RNA’s need to be degraded - homeostatis and quality control?

Answer 1

Some are bi-products of transcription and need to be removed and resources recycled
They weren’t made correctly and could damage cell if made into a protein

Question 2

Why do RNA’s need to be degraded? - gene regulation

Answer 2

If they never went away how could you have gene regulation
Highly regulated mRNA’s tend to have short half lives

Question 3

How do exonucleases degrade RNA?

Answer 3

From the end 3’ - 5’ or 5’ - 3’

Question 4

How do endonuclease degrade RNA?

Answer 4

Internally

Question 5

What is the major human 5’ - 3’ exonuclease present in the nucleus?

Answer 5

Xrn2

Question 6

What does xrn2 degrade?

Answer 6

Transcriptional termination of Pol II and pre-mRNA degradation (when splicing is disturbed or decapping has occurred).

Question 7

Where does xrn2 degrade?

Answer 7

Cytoplasm

Question 8

What needs to happen before xrn2 can degrade?

Answer 8

Decapping

Question 9

What is the major 3’ - 5’ exonuclease activity in the nucleus an cytoplasm?

Answer 9

The exosome

Question 10

What is the exosome involved in?

Answer 10

Processing and degradation

Question 11

Where is most RNA degraded in?

Answer 11

The nucleus

Question 12

When can RNA be degraded?

Answer 12

During or after its synthesis

Question 13

Do introns need to be degraded?

Answer 13

Yes

Question 14

What happens in the quality control pathway after the start of RNA transcription?

Answer 14

Possible attenuation, capping, splicing and 3’ end cleavage, possible RNA editing and nuclear export

Question 15

What happens when RNA transcriptions goes wrong after the start of transcript?

Answer 15

RNA transcript aborts, nonfunctional mRNA sequences, retention and degradation in the nucleus.

Question 16

See other cue cards

Question 17

Degradation of proper mRNA’s - what do you need to do to do this?

Answer 17

Get rid of polyA tail (deadenylase) and 5’ cap

Question 18

What happens first poly A tail degradation or the cap?

Answer 18

PolyA degradation

Question 19

Is the polyA tail in the proximity of the 5’ cap?

Answer 19

Yes

Question 20

Regulated turnover - what is this?

Answer 20

mRNA was made properly but it is no longer needed

Question 21

What is a half life?

Answer 21

The time it takes for half of a message to go away

Question 22

The untranslated region - Why is the 3’ UTR so important?

Answer 22

Encodes regulatory features, RNA binging proteins can bind here as can microRNA binding sites

Question 23

What can affect deadenylation if the 3’ UTR?

Answer 23

Specific proteins and specific RNPs

Question 24

How are RNA’s recognised by specific proteins?

Answer 24

Through contact or through proteins recognising sequences

Question 25

Where are ARE-mediated mRNA found?

Answer 25

In the 3’UTR

Question 26

What recognises the ARE?

Answer 26

Proteins which stabilise (HUR) and destabilise (TTP) the mRNA

Question 27

Non-ARE mediated mRNA can also be involved with stabilising and destabilising of mRNA in the 3’UTR what are these and do they stabilise or destabilise?

Answer 27

UCR elements are recognised by PTB which stabilises

Question 28

Stabilisation/destabilisation of mRNA via 3’UTR = how does miRNA- mediated mRNA happen?

Answer 28

MiRNA regulators work in a risc complex. MiRNA binds to argonot which takes it to the mRNA promoting deadenylation.

Question 29

What are the ARE’s which destabilise mRNA?

Answer 29

TTP, KSRP and AUF

Question 30

What are the ARE’s which stabilises mRNA?

Answer 30

HUR and sometimes AUF

Question 31

The factors which stabilise or destabilise mRNA aren’t regulated by signaling, true or false?

Answer 31

False - they are

Question 32

How do the stabilising or destabilising factors work?

Answer 32

By recruiting exonucleases

Question 33

microRNA regulation of mRNA stability - how are these generally transducer?

Answer 33

As pol II transcripts found in introns

Question 34

microRNA regulation of mRNA stability - what do these bind to?

Answer 34

Complementary sequences

Question 35

Reporter assays - how are these done - step 1: promoter insertion and reporter

Answer 35

You add a promoter you are interested from your mRNA to a reporter gene (this is not your mrna). You would put it either upstream or downstream of the reporter depending on where the promoter is

Question 36

Reporter assays - how are these done - step 2: transfect

Answer 36

You transfect it into cells and incubate it until protein is formed. You then measure the protein or the mRNA over different timeframes

Question 37

Why do we use reporter assays?

Answer 37

To test the importance/ regulation of mRNA in different contexts e.g. we can see if a gene is getting down regulated.

Question 38

What are the controls of the reporter assay?

Answer 38

Transfection of another reporter to make sure you know how much reporter you are getting in.

Question 39

What are the requirements of a reporter assay in terms of something that can be quantified?

Answer 39

Luciferase, GFP and lacZ

Question 40

What is a caveat of reporter assays?

Answer 40

Sometimes the reporter does not integrate into the native location of the gene in the genome and therefore you can’t see any interesting effects. Cell lines should also be mimicked in its natural environments

Question 41

How do you measure promoter activity?

Answer 41

Reporter assay by making numerous deletions upstream of the start site and put each into the reporter plasmid turning it into DNA. You then transfect it into a eukaryotic cell. You can then see what important for transcription

Question 42

When infected the promoter of luciferase drops and when you stop transcription the promoter of luciferase also drops - what does this mean?

Answer 42

When transcription stops luciferase promoter drops meaning that infection stops transcription.

Question 43

What would you expect to see if the RNA had a long half life after turning off transcription?

Answer 43

Rna still being present

Question 44

How do you measure the half life of a mRNA?

Answer 44

Stop transcription and measure how long the mRNA lasts

Question 45

How do you stops transcription?

Answer 45

Inhibitors to block

Question 46

What inhibitors block transcription?

Answer 46

Actinomycin D Alpha-amanitin

Question 47

How does actinimycin D strop transcription?

Answer 47

intercalated into DNA and blocks transcription and elongation

Question 48

How does alpha-amanitin stop transcription?

Answer 48

Binds to subunit of RNA pol II

Question 49

The steps of measuring half life of mRNA?

Answer 49

Add a inhibitor to stop transcription at time 0. Then harvest rna at different time points and measure it. Make a graph and determine the point at which you have half the amount of mRNA than you started with

Question 50

What is the caveat to using inhibitors to block transcription?

Answer 50

They are toxic to a cell

Question 51

What effects the half life of mRNA?

Answer 51

Elements in the UTR