Lecture 21 - RNA export and localisation, RNA decay

Question 1

What are the features of RNA export from the nucleus to the cytoplasm?

Answer 1

• nucleo-cytoplasmic transport occurs through nucleur pores
• nuclear pores span the nuclear membrane
• composed of nuclear porin proteins
• have a centreal channel through which RNA and proteins can pass
• 1000s in the nuclear membrane
• only very small molecules can diffuse through
• larger, e.g. RNA must be actively brought throught

Question 2

In the nuclues, mRNAs exist as large RNA protein complexes - what are the proteins assosicated?

Answer 2

• Cap binding and polyA binding proteins
• General RNA binding proteins
• proteins deposited during splicing
• RNA export factors

Question 3

What is necessary to help mRNA leave the nucleus?

Answer 3

mRNA export receptor

Question 4

What is the structure and function of the mRNA export receptor?

Answer 4

2 main proteins involved:

• Mex67 (yeast), Tap (metzoans)
• Mtr2 (yeast) p15 (metazoans)
• Mex67 and the Mtr2 dimerise and interact with the mRNA via adapter proteins and with nuclear porins that line the nuclear pore
• RBD, LRR, M, C
• LRR involved in protein:protein interactions

Question 5

When are mRNA export receptors recruited?

Answer 5

• during a nuclear processing event and only to correctly processed mRNAs
• in yeast can’t be recruited in splicing because most yeast genes have no introns

Question 6

What is the process of transcription coupled export in yeast?

Answer 6

• TREX proteins deposited on mRNA allows export receptor to be recruited onto the mRNA
• cap binding proteins are also involved
• exchange between export receptor and TREX leads to 2 subunits of TREX being knocked off, one remains
• followed by tranlocation and termination

Question 7

What is the process of splicing coupled RNA export in metazoans?

Answer 7

• TREX recruited to mRNA
• the process of splicing actually promotes export proteins landing on the part that is triggering the recruitement of TREX1
• cap binding proteins are also involved (present earlier than in yeast)
• there is a deposition of proteins at the exon junction
• this is involved in recruiting export receptors (via which method vaires between different RNAs)
• 2 TREX subunits are removed and 1 remaining (due to export receptors/translocation

Question 8

What are the key events in RNA export?

Answer 8

• RNA binding proteins are recruited to mRNAs during RNA pol II transcription, splicing, capping and polyadenylation
• export receptors are recruited via adapter proteins and some can be depositied by TREX
• export receptors interact with the nuclear pore
• remodelling of mRNA follows export

Question 9

What is a key mechanism to allow for asymmetric distibution of cytoplasmic factors?

Answer 9

-local restriction of protein synthesis is via localisation of mRNAs

Question 10

What are the features of cytoplasmic RNA localisation?

Answer 10

• local restriction of protein synthesis via localisation of mRNAs
• localisation is found in a wide variety of cell types and organisms (not just higher eukaryotes)
• it is estimated that in yeast 1% of transcripts are localised
• in drosophila it is possible that 70% may be localised (viewed by in situ hybridisation, embryos strictly localised (segments))

Question 11

Give three examples of how RNA can be localised to target individual gene products to specific regions

Answer 11

• High concentration of proteins at the leading edge of fibroblasts
• gradient of morphogens e.g. bicoid in drosophila
• association with specific subcellular structures e.g. mitotic apparatus

Question 12

What are the three possible mechanisms of RNA localisation?

Answer 12

1. direct transport via the cytoskeleton
2. random diffusion then mRNAs are trapped where required
3. generalised degradation combined with local protection - mRNAs unstable apart from in the area they need to be localised to

Question 13

How can direct transport of mRNA localisation via the cytoskeleton be imaged?

Answer 13

mRNA labelled in vivo with GFP

• tubulin labelled in red
• mRNA seen tracking along a microtubule

Question 14

Where does the information come from that defines where the mRNA will be localised?

Answer 14

mRNA localisation signals on the mRNA

-sequence within the 3’UTR that directs localisation

Question 15

Why is mRNA localised before being translated and not the other way around? (moving proteins)

Answer 15

• protein may ACT before it can be moved, aka in an area you don’t want it to act
• also would need localising information in the protein sequence itself
• multiple proteins must share the same localisation code but by localising mRNA don’t need to share the same aa sequence

Question 16

When does mRNA decay?

Answer 16

after translation

Question 17

What is more stable, eukaryotic mRNA or prokaryotic mRNA?

Answer 17

Eukaryotic mRNA

Question 18

How is eukaryotic mRNA protected against the activity of exonucleases?

Answer 18

• processing events have modified the 5’ end and 3’ ends (Cap binding proteins and polyA binding at either end)
• these interact with translation factors and form a circular structure
• this structure has impacts in translation and stability

Question 19

What are the varios pathways for mRNA decay in eukaryotes?

Answer 19

Deadenylation dependent 
-remove the polyA tail first
-most common
Deadenylation independent 
-don't need to remove polyA tail first 
Endoribonucleases
-endonuclease cuts in the middle then degrade from that point
Non-sense mediated decay 
-used on aberant mRNA when not perfect

Question 20

What are the enzymes needed for deadenylation dependent RNA decay?

Answer 20

Enzymes needed:
-CCR4, CAF1, PARN (deadenylation)
3'-5' decay
-Exosome (3'-5' decay)
-DcpS (3' - 5' decapping)
OR 5'-3' decay
-Dcp1 and Dcp2 (5'-3' decapping)
-Xml (5'-3' decay)

Question 21

What is the process of deanylation dependent RNA decay?

Answer 21

-RNA is accessible to an extent, not a covelently bound circle
-once the polyA tail has been removed by (CCR4, CAF1, PARN) the remainder is degraded in one of two ways:
-exosome continues degradation in the 3’to 5’ direction then DcpS removed the 5’ cap
OR
-decapping occurs first via (Dcp1 and Dcp2) then degradation by XrnI in the 5’ to 3’ direction

Question 22

What are important features of RNA decay?

Answer 22

• all mRNAs undergo RNA decay and have an inherant level of stability
• RNA stability can be regulated
• unstable mammallian mRNAs often contain A and U rich elements (AREs) in the 3’ untranslated region

Question 23

How do AREs cause instability?

Answer 23

• AU binding proteins recognise the AU rich regions and interfere with the circularisation structure and how tightly other proteins can interact
• this results in a looser interaction, so mRNA is more accessible to enzymes (and degradation)

Question 24

Which mRNAs would be intentionally unstable?

Answer 24

those involved in the cell cycle as they are only required for a short period of time

Question 25

What are the features of non-sense mediated decay?

Answer 25

- ensures that mRNAs with premature termination codons are eliminated as templated for translation - necessary as trunctated proteins can have a dominant negative effect

Question 26

How is it known that an mRNA contains a premature stop codon in nonsense mediated decay?

Answer 26

Mammals - splicing - genes rarely have introns in the 3' UTR - the stop codon is usually in the last exon of the ORF - in there is an intron in the 3' UTR it is always less than 5-nts downstream of the stop codon - therefore splicing is very important - insertion of an intron more than 50bp downstream of the last exon and stop codon, that mRNA is targeted for nonsense mediated decay, as it suggests that stop codon is actually premature

Question 27

What evidence is there that translational machinery is involved in nonsense mediated decay?

Answer 27

- cis mutations that prevent assembly of ribosomes on the PTC-containing mRNAs can stabilise the transcripts - PTC-containing transcripts are associated with polysomes - drugs that inhibit translation (e.g. cycloheximaide) can stabilise nonsense-containing mRNAs

Question 28

How are exon junction complexes involved in nonsense mediated decay?

Answer 28

- depositied 20-24nts upstream of the exon exon junction during splicing - EJC contains at least 5 proteins, some of which remain attached to the RNA in the cytoplasm (not all are remodelled during the process of RNA export)

Question 29

What UPF proteins are involved in nonsense mediated decay?

Answer 29

Upf1, Upf2, Upf3 - interact with each other - Upf1 interacts with translation termination factors - Upf3 interacts with the exon junction complexes - forms a bridge between the position of the stop codon and EJC

Question 30

What happens to EJCs in regular and PTC-containing mRNAs?

Answer 30

Normal -all EJCs are removed by the translocating ribosome when first translated PTC-containing mRNA -as the ribosome is translating, the UPF proteins form a bridge with the premature stop codon and a downstream EJC -EJC should NOT be downstream of stop codons -triggers the degradation of mRNA initially though decapping and then through 5' to 3' exonuclease activity

Question 31

How do yeast determine whether a stop codon is premature or not? (Nonsense mediated decay)

Answer 31

Translational protein binds to the 3' untranslated region and judges the distance between the stop codon -length normally highly conserved

Question 32

What is involved in the initial round of translation?

Answer 32

- newly exported mRNAs have an RNP (ribonucleoprotein) structure distinct from the mature RNAs - may be an initial round of translation that is different from subsequent translation - during the inital round of translation, nuclear proteins that were bound during mRNA maturation (e.g. EJCs depositied during splicing) are removed - during translational termination a mechanism is in place that scans for downstream EJC complexes and triggers RNA degradation if the transcript is considered abbrrant May be a quality control mechanism

Question 33

Give an example in nature of how premature stop codons are quality control mecahnisms

Answer 33

in drosophila sex determination pathway -sxl regulated alternative splicing of its own pre-mRNA -in males the sxl transcript has a premature stop codon, forms unfunctional SXL protein Doesn't happen in females

Question 34

How can nonsense mediated decay have an effect on disease phenotypes?

Answer 34

e.g. Thalassaemia Human β-globin mRNA carries a PSC in thalassamias -3 exons -if premature stop codon is in Exon 2 then nonsense mediated decay is triggered (exon:exon boundary, EJC, stop codon, Upf interactions) and the truncated protein is not produced, mutations are recessive -if the premature stop codon is in Exon 3 then there no is nonsense mediated decay (no downstream EJC) and the truncated protein is produced and mutations are dominant, show a dominant negative effect