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mRNA decay- regulating gene expression -

Wiebke Ginter06.12.10

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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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Post-transcriptional downregulation by non-coding RNAs

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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

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The End