Photography and the Archive Research Centre - Fieldstudy 11 - Wiebke Leister
1 mRNA decay - regulating gene expression - Wiebke Ginter 06.12.10.
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Transcript of 1 mRNA decay - regulating gene expression - Wiebke Ginter 06.12.10.
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Differences of eukaryotic and bacterial mRNA
Bacterial mRNA
- Triphosphate
- Stem-loop
- Ribosome binding: base pairing between the 3’ end of 16S ribosomalRNA and a Shine–Dalgarno element
Eukaryotic mRNA
-5’ 7-methylguanosine cap
-3’ poly(A) tail with poly(A)-binding protein (PABP)
-Ribosome binding: affinity of the small ribosomal subunit for eukaryotic initiation factor 3 (eIF3)
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Conventional pathways for mRNA degradation (E. coli)
- serial internal cleavage by RNase E
- lack base pairing at the 3’ end
- susceptible to attack by the 3’ exonucleases polynucleotide phosphorylase (PNPase), RNase II, RNase R and oligoribonuclease
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Conventional pathways for mRNA degradation (Eukaryotes)
PAN2-PAN3• PABP-dependent poly-A
nuclease, 60-80nt
CCR4-NOT• 9 protein• exonuclease domains in Ccr4
and Caf1• activity inhibited by PABP
PARN• Cap-dependent deadenylase• processivity enhanced by
5’cap• inhibited by cap-binding
proteins• mass deadenalytion in
maternal mRNA in oocytes (Xenopus), in various cell lines, embryogenesis in plantsDcp1/2
• Decapping enzyme• dimer
XRN1 • exoribonuclease• degrades 5′→3′ direction
Exosome• Large complex of 3′→5′ exonucleases• 10-12 SU with RNase PH domain• homologies with hydrolytic exonucleases, RNA helicases
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P-bodies
Lsm1 XRN1 DNA
- Cellular sites of decay, but also RNA storage
- Granular cytoplasmic foci
- Enriched in components of 5’ → 3’ decay
- assemble when 5’ → 3’ decay system is overloaded with mRNA or decay is impaired
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Unusual routes to decay
Deadenylation-independent decapping
- bypass deadenylation step – directly decapped
- autoregulatory
- Rps28B directly binds stem-loop of 3’ UTR of own mRNA
- recruits Edc3 – enhancer of decapping
- association of other decapping factors
Edc1: decapping regulatorintramolecular pairing blocks access to the deadenylase: interaction between the poly(A) tail and a poly(U) stretch in the 3′ UTRfeedback regulation
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Unusual routes to decay
Endoribonucleolytic decay
- PMR: polysome-associated endonuclease
- Targeting actively translating mRNA
- IRE1: endonuclease on endoplasmic reticulum
- Targeting actively translating mRNA
- MRP: multicomponent complex, RNase
- Processing rRNA/nucleolus, mitochondrial RNA
- In temporal asymmetric MRP bodies during mitosis
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Non-sense mediated decay (NMD) - I
• Detects premature termination codons (PTC)
• arise from mutations, frame-shifts, inefficient processing, leaky translation initiation and extended 3’ UTR
• truncated proteins with aberrant functions
• Core proteins of the NMD complex: UPF1, UPF2 and UPF3
• exon junction complex (EJC)• feature of an aberrant
transcript, residual ‘mark’ of splicing
• 20–24 nucleotides upstream EJ
• Also role in regulating normal gene expression
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Non-sense mediated decay (NMD) - II
Most: deadenylation-independent decapping in P bodies
• Detects premature termination codons (PTC)
• arise from mutations, frame-shifts, inefficient processing, leaky translation initiation and extended 3’ UTR
• truncated proteins with aberrant functions
• Core proteins of the NMD complex: UPF1, UPF2 and UPF3
• exon junction complex (EJC)• feature of an aberrant
transcript, residual ‘mark’ of splicing
• 20–24 nucleotides upstream of every
• Also role in regulating normal gene expression
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Non-stop decay
• Targets mRNAs that lack a stop codon
• Premature polyadenylation
• facilitates the release of the ribosome
• Ski-complex (Ski1,3,8)• Ski7 (adaptor) binds to
empty A site• release ribosome• Ski7 recruits exosome• SKI-complex
deadenylates• decay 3’→5’ direction
• No Ski7: 5’ → 3’ decay pathway (due to PABP removal)
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No-go decay
• Detecting stalled ribosomes
• Endonucleolytically cleaving the mRNA
• Dom34-Hbs1 needed for initial cleavage
• decayed by the exosome and Xrn1
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Signals that control mRNA decay
AU-rich elements (ARE)
● Stability element● 3’UTR of cytokines, proto-
oncogenes, transcription factors● AUUUA-pentamer – several classes● No 2 identical● Flanking region can influence
overall effect on mRNA stability ● Enhance decay by recruiting
mRNA-decay machinery● Interacts with exosome (AUF1, TTP)● Bind PARN deadenylases (KSRP,
RHAU)
Stabilising mRNA-binding proteins
● Removing mRNA from decay sites?● Competing with binding sites for
decay factors?● Inhibit decay machinery?● Strenghten PABP-poly(A)
interaction?
Modulation of RNA-binding proteins● mRNA=unstable=facilitate rapid
changes if mRNP is changed● P38 MAPK, ERK, JNK, Wnt/β-catenin
pathways influence ARE-function● Modulate mRNP structure, mediate
phosphorylation of ARE-binding proteins, alter affinity, bind other factors
Puf proteins● Recognise UG-rich sequences● Accelerates decay● CCR4-NOT deadenylase recruited● Each Puf has special target
transcripts● Regulate certain cellular processes
Stabilising elements● Sequence elements can confer
stability = transcripts of housekeeping proteins = stable
● Pyrimidine-rich elements in 3’ UTR● αCP1 and αCP2 bind● Protecting poly(A) tail from
deadenylation
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Interfacing with other cellular mechanisms
Translation● General inhibitions of translation elongation → stabilising mRNAs on
polysomes● Inhibtition of translation initiation → diverts transcripts to P-bodies for decay● Many mRNA-binding proteins that influence mRNA turnover also regulate
translation
Transcription● CCR4–NOT complex represses RNA polymerase II required for both
transcription and deadenylation ● Rpb4 protein
- subunit of RNA polymerase II- also required for deadenylation and decay- localizes to P bodies- essential role in modulating gene expression in response to stresses
such as glucose deprivation and heat shock
mRNA localisation● DCP1 and CCR4 – implicated in localisation of mRNA transcripts
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Key differences mRNA decay
Bacterial decay
• Transient addition of poly(A) tails=crucial for exonucleolytic degradation of stem-loop structures
• Pyrophosphohydrolase: conversion of 5’ terminal triphosphate to monophosphate
→ more susceptible to 5‘ monophosphate dependent RNase=endonuclease RNase E
• Quality control: PTC
• Non-stop decay: tmRNA
• No-go decay: endonuclease
Eukaryotic decay
• Poly(A) tail
• Resemblance to decapping (catalyses by related enzymes)=removing a protective group
→ more susceptible to 5‘ monophosphate dependent RNase=exonuclease XRN1
• Quality control: PTC, recognise abnormal 3’ UTR
• Non-stop decay: Ski7
• No-go decay: endonuclease
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Key differences mRNA decay
Bacterial decay
• Mostly by low specificity endonucleases
• Poor ribosome binding → decay (spacing increases, cleavage sites free)
• Shorter intercistronic and 3’ UTR
• Poly(A)= destabilising
• Internal ribosome binding sites – co-transcribed polycistronic operons possible – can also degrade discrete segments only
Eukaryotic decay
• 3’ and 5’ terminal events=dominant (deadenylation, decapping, exosomes)
• Endonucleases=much less, more specific
• Inefficient initiation: not doomed to degradation
• 3’ UTR long, contains binding sites for regulating proteins
• Depend on deadenylation of stabilising poly(A)– need of protective PABP
• eIF4F protein complex governs terminal ribosome binding, interaction with PABP and poly(A) tail interupted by deadenylation