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Gene Expression > Coupling of transcription and RNA decay > Flashcards

Coupling of transcription and RNA decay Flashcards

1
Q

What are we traditionally taught about gene expression in eukaryotes?

A

Transcription and RNA decay are independent events.

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2
Q

How many factors impact the fate of an mRNA?

A

7

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3
Q

How does polyA tail length impact the fate of an mRNA?

A

(In eukaryotes) transcripts with longer polyA tails are less susceptible to degradation because the 3’ end is protected.

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4
Q

How does splicing rate impact the fate of an mRNA?

A

Slow splicing = more frequent degradation. E.g. ribosomal proteins are spliced quickly and not degraded.

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5
Q

How does rate of nuclear export impact the fate of an mRNA?

A

Slow export = more frequent degradation.

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6
Q

How does RNA sequence impact the fate of an mRNA?

A

Specific sequences recruit RBPs e.g. which help recruit decay machinery to RNA. Often in the 3’ UTR.

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7
Q

How does RNA structure impact the fate of an mRNA?

A

Highly structured mRNAs tend to have longer half-lives e.g. stable stem loop at the beginning or end blocks nucleases.

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8
Q

How does codon sequence impact the fate of an mRNA?

A

mRNAs with many rare codons are not efficiently translated due to low frequency of these tRNAs; get degraded faster as ribosome pausing triggers decay.

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9
Q

How do ncRNAs impact the fate of an mRNA?

A

ncRNAs. miRNAs, bacterial sRNAs. Act in cis or in trans. Bind 3’ UTR and dictate translation efficiency and decay factor recruitment.

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10
Q

What are we traditionally taught about RNA decay in eularyotes?

A

Specific factors (RBPs) bind to sequences in the 3’ UTR, and they recruit or block nucleases ie promote or block decay.

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11
Q

What are new theories about RNA decay pointing to?

A

The fate of the mRNA can be dictated by cis-acting elements that are NOT part of the mRNA sequence (only the DNA).

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12
Q

What are theories coupling transcription to degradation?

A
  • Polymerase subunits assist in mRNA degradation in the cytoplasm.
  • The promoter sequence can determine the half-life of an mRNA.
  • Transcription factors recruit RNA-binding proteins to mRNAs that regulate the decay of the mRNA in the cytoplasm.
  • Transcription factor itself binds to mRNAs that regulate the decay of the mRNA in the cytoplasm.
  • Promoter elements can determine the sub-cellular localisation of the mRNA and how well it is translated.
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13
Q

What are the mechanisms of nuclear and cytoplasmic RNA decay in eukaryotes?

A
  • Nuclear RNA decay
  • Cytoplasmic RNA decay (cytoplasmic decay factors and P bodies and stress bodies).
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14
Q

How does mRNA circularise?

A

The eIF cap binding complex interacts with the Pab1 proteins bound to the polyA tail.

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15
Q

Why does mRNA circularise?

A

To make translation circular; makes it easier for ribosomes to reinitiate translation by bringing them into close proximity with the promoter when they dissociate. Also allows cell to distinguish between faulty and correct mRNAs.

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16
Q

How is circular mRNA targeted for degradation?

A

The polyA tail is digested by specific exosomal enzyme complexes (Pan2/3 and Ccr4/Not), which displaces Pab1.

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17
Q

How is circular mRNA degraded?

A

Trimming of the polyA tail and Pab1 displacement allows:
- Lsm1-7 and Pat1 recruitment to the 3’ end.
- Recruitment of Dcp2 and its cofactors to decap the 5’ end.
- Xrn1 exonuclease degradation (5’ to 3’).
- Exosome complex degradation (3’ to 5’).

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18
Q

What is the exosome?

A

A highly conserved complex found in the nucleus and cytoplasm of eukaryotes (also has an ancestor in archaea). It degrades mRNA in the 3’ to 5’ direction.

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19
Q

What is the ski complex?

A

A cytoplasmic protein complex that associates with the exosome and helps recruit it to deadenylated mRNAs. Ski2p has helicase activity so removes RNA folding that would prevent degradation as the RNA passes through it into the core complex.

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20
Q

What is the core exosome complex?

A

A protein complex present in all exosomes that catalyses 3’ to 5’ phosphorolytic decay of RNA. Related to bacterial PNPase.

21
Q

What are the components of the core exosome complex?

A
  • S1 / KH cap.
  • PH ring (hexameric).
  • Dis3p.
22
Q

How does the core exosome complex function?

A

S1/KH cap guides RNA through the PH ring, which directs the RNA to the Dis3p which degrades the RNA.

23
Q

Why is exonucleolytic degradation thought to be the predominant pathway (rather than endonucleolytic)?

A
  • Strains lacking decapping enzyme are very sick (lethal in some backgrounds)
  • Strains lacking decapping or 5’-3’ degradation show elevated steady state levels and decreased decay rates of MANY RNAs
  • Genome-wide mapping of endonuclease sites reveals they are present in very few RNAs.
24
Q

Do degradation and translation happen at the same time?

A

Yes - Xrn1 degrades as ribosome is translating.

25
Q

How do we map the location of ribosomes on an mRNA as it is being degraded?

A

Through purifying the products of Xrn1 mediated degradation.

26
Q

What are P bodies composed of?

A

mRNAs and highly disordered mRNA binding proteins that are involved in translation repression and mRNA decay.
No membrane!

27
Q

What is the current theory of P body function?

A

mRNA storage; the mRNAs can exit and return to translation e.g. when the cell envounters an environment better for growth.
Some people think it is the site of mRNA due to the high concentration of decay factors.

28
Q

What are stress granules composed of?

A

mRNAs and translation initiation factors. Interactions between intrinsically disordered regions of proteins.
No membrane!

29
Q

What is the current theory of stress granule function?

A

mRNA storage - represses translation of the mRNAs within it.

30
Q

When do stress granules and P bodies tend to form?

A

When a cell is under stress; essential for cell survival under stress. Highly conserved in eukaryotes.

31
Q

What is a docked granule?

A

A P body and a stress granule bound to each other. They are able to exchange components.

32
Q

How are transcription and RNA decay coupled?

A

The transcription machinery is not only responsible for transcript synthesis, but also determines how quickly it is degraded.

33
Q

What is transcription machinery?

A
  • RNA pol II
  • Transcription factors
34
Q

What is an incoherent feed forward loop (iFFL)?

A

The transcription machinery facilitates transcription, but also induces the expression of or recruits components (ncRNA or RBP) that initiate mRNA decay or decrease transcript stability.

35
Q

What is a coherent feed forward loop (cFFL)?

A

The transcription machinery facilitates transcription and also induces expression of or recruits components that enhance translation or increase mRNA stability.

36
Q

What is the point of an iFFL?

A

The translation inhibitors / decay machinery have to accumulate before they can reduce mRNA. Only once they have reached a ‘critical level’ do they impact mRNA levels. This allows a ‘pulse like’ expression response.
The expression of only select genes controlled by one TF can be controlled.

37
Q

What is a ‘pulse like’ expression response?

A

Once the mRNA has reached ‘critical level’ it drops quickly (shorter ‘response time’). This allows shorter gene expression pulses. This is important in development and environmental response.

38
Q

Why do ncRNAs and RBPs not impact mRNA levels until the ‘critical level’ is reached?

A

They act on many mRNAs so do not have a significant impact on a single one.

39
Q

What is the point of a cFFL?

A

It can increase the speed at which protein levels change in response to a stimulus.

40
Q

What is thought to be the function of the Rpb4/Rpb7 heterodimer?

A
  • Has a role in active transcription (associates with RNA pol II).
  • Associates with the nascent transcript as it exits the nucleus and accumulates in the cytosol.
  • Role in localising nacsent mRNAs to P bodies (Rpb4 deletion = P body mRNA accumulation) and therefore dictating mRNA half lives / decay.
  • mRNAs bound by it are more likely to be translated once the stress has passed (?)
41
Q

How did the Choder lab demonstrate Rbp4/Rpb7 exit the nucleus and accumulate in P bodies under stress?

A

GFP was used to show that after 4 hours of starvation the Rpb4 (green fluorescence) localised to the P bodies (colocalises with Dcp2) instead of the nucleus. A yeast two-hybrid screen to show Rpb4 physically interacts with Lsm2 and Pat1 cytoplasmic decay proteins (and Rpb7). This is stronger evidence than the fluorescence which is a bit tenuous.

42
Q

What is the iFFL that has been proposed for mRNA transcription regulation?

A

Pol II promotes transcription but also the release of Rpb4/7 which (in normal conditions) stimulates RNA decay via recruitment of Pat1 and subsequent decay machinery.

43
Q

Why is the Rpb4/7 function not clear?

A
  • Claims that an mRNA with an Rpb4/7 tag is destined to be degraded (decay machinery association).
  • Claims that an mRNA with an Rpb4/7 tag is destined to be stored and released (P body localisation).
44
Q

What is the cFFL that has been proposed for mRNA transcription regulation?

A

In stress conditions, the Rpb4/7 actually promotes translation by localising it to P bodies so it can be expressed when cellular stress is over.

45
Q

Can promoter sequences contribute to mRNA decay in the cytoplasm?

A

Yes -

46
Q

What did the O’Shea lab discover about gene expression in response to glucose starvation?

A

Heat shock proteins (stress response) had increased mRNA and translation levels, whereas glycolysis proteins had increased mRNA but decreased translation levels. Ribosomes were held at the 5’ end of the transcripts in glycolysis mRNAs.

47
Q

What is the current model for how the glycolysis mRNAs upregulated but not translated?

A

They are localised to P bodies via a cFFL. Their promotor is bound by a TF that recruits Rvb1/2, which is exported promotes transcription but once it is exported to the cytoplasm tags the transcript to be stored in P bodies. (Could argue against cFFL because translation is not enhanced). This primes the cell for the return of glucose levels (bet hedging).

48
Q

What is the current model for how heat shock protein translation is upregulated under cellular stress?

A

The heat shock elements (HSEs) in the heat shock protein promoter is bound by Hsr1 TF. This promotes translation and also recruits an RBP which further promotes translation (cFFL) by binding the mRNA and blocking RNA decay factors.

49
Q

How can you identify differences in proteins binding plasmids with different promoters?

A

Synthesise plasmids with the same ORF but different promoters. Purify plasmids using a flag tagged LacI that will bind a LacO in the plasmid. Use mass spec to identify proteins enriched for these plasmids in a promoter specific manner.

Gene Expression (9 decks)

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