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Molecular Genetics > Lecture 6 - Regulatory Mechanisms For Quality Control And Turnover Of Pre-mRNA > Flashcards

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

1
Q

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

A

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

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2
Q

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

A

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

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3
Q

How do exonucleases degrade RNA?

A

From the end 3’ - 5’ or 5’ - 3’

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4
Q

How do endonuclease degrade RNA?

A

Internally

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5
Q

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

A

Xrn2

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6
Q

What does xrn2 degrade?

A

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

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7
Q

Where does xrn2 degrade?

A

Cytoplasm

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8
Q

What needs to happen before xrn2 can degrade?

A

Decapping

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9
Q

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

A

The exosome

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10
Q

What is the exosome involved in?

A

Processing and degradation

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11
Q

Where is most RNA degraded in?

A

The nucleus

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12
Q

When can RNA be degraded?

A

During or after its synthesis

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13
Q

Do introns need to be degraded?

A

Yes

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14
Q

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

A

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

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15
Q

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

A

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

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16
Q

See other cue cards

A
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17
Q

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

A

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

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18
Q

What happens first poly A tail degradation or the cap?

A

PolyA degradation

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19
Q

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

A

Yes

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20
Q

Regulated turnover - what is this?

A

mRNA was made properly but it is no longer needed

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21
Q

What is a half life?

A

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

22
Q

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

A

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

23
Q

What can affect deadenylation if the 3’ UTR?

A

Specific proteins and specific RNPs

24
Q

How are RNA’s recognised by specific proteins?

A

Through contact or through proteins recognising sequences

25
Q

Where are ARE-mediated mRNA found?

A

In the 3’UTR

26
Q

What recognises the ARE?

A

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

27
Q

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?

A

UCR elements are recognised by PTB which stabilises

28
Q

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

A

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

29
Q

What are the ARE’s which destabilise mRNA?

A

TTP, KSRP and AUF

30
Q

What are the ARE’s which stabilises mRNA?

A

HUR and sometimes AUF

31
Q

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

A

False - they are

32
Q

How do the stabilising or destabilising factors work?

A

By recruiting exonucleases

33
Q

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

A

As pol II transcripts found in introns

34
Q

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

A

Complementary sequences

35
Q

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

A

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

36
Q

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

A

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

37
Q

Why do we use reporter assays?

A

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

38
Q

What are the controls of the reporter assay?

A

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

39
Q

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

A

Luciferase, GFP and lacZ

40
Q

What is a caveat of reporter assays?

A

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

41
Q

How do you measure promoter activity?

A

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

42
Q

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

A

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

43
Q

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

A

Rna still being present

44
Q

How do you measure the half life of a mRNA?

A

Stop transcription and measure how long the mRNA lasts

45
Q

How do you stops transcription?

A

Inhibitors to block

46
Q

What inhibitors block transcription?

A

Actinomycin D
Alpha-amanitin

47
Q

How does actinimycin D strop transcription?

A

intercalated into DNA and blocks transcription and elongation

48
Q

How does alpha-amanitin stop transcription?

A

Binds to subunit of RNA pol II

49
Q

The steps of measuring half life of mRNA?

A

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

50
Q

What is the caveat to using inhibitors to block transcription?

A

They are toxic to a cell

51
Q

What effects the half life of mRNA?

A

Elements in the UTR

Molecular Genetics (20 decks)

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