Transcript Efficient Immunization by Targeting Human DC

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 ribosomal
RNA 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
Dcp1/2
• Decapping enzyme
• dimer
XRN1
• exoribonuclease
• degrades 5′→3′ direction
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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 plants
Exosome
• Large complex of 3′→5′ exonucleases
• 10-12 SU with RNase PH domain
• homologies with hydrolytic exonucleases, RNA
helicases
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
Deadenylationindependent 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
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Edc1: decapping regulator
intramolecular pairing blocks access to the
deadenylase: interaction between the poly(A) tail
and a poly(U) stretch in the 3′ UTR
feedback regulation
Unusual routes to decay
Endoribonucleolytic decay
- PMR: polysome-associated
endonuclease
- Targeting actively translating
mRNA
- IRE1: endonuclease on
endoplasmic reticulum
- Targeting actively translating
mRNA
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- MRP: multicomponent complex,
Non-sense mediated decay (NMD) - I
• Detects premature termination
codons (PTC)
• arise from mutations, frameshifts, 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
• Detects premature termination
codons (PTC)
• arise from mutations, frameshifts, 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
Most: deadenylation-independent decapping in P bodies
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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 mRNAdecay 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?
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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
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
Eukaryotic decay
•
•
Poly(A) tail
•
Resemblance to decapping (catalyses
by related enzymes)=removing a
protective group
•
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=exonuclease XRN1
→ more susceptible to 5‘ monophosphate
dependent RNase=endonuclease
RNase E
•
Quality control: PTC, recognise
abnormal 3’ UTR
•
Quality control: PTC
•
Non-stop decay: Ski7
•
Non-stop decay: tmRNA
•
No-go decay: endonuclease
•
No-go decay: endonuclease
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Key differences mRNA decay
Bacterial decay
Eukaryotic 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 – cotranscribed polycistronic operons
possible – can also degrade discrete
segments only
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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
The End
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