mRNA decay - regulating gene expression -

1
mRNA decay- regulating gene expression -
Wiebke Ginter06.12.10
2
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 ShineDalgarno 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)

3
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

4
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 5cap
• inhibited by cap-binding proteins
• mass deadenalytion in maternal mRNA in oocytes
  (Xenopus), in various cell lines, embryogenesis
  in plants

• Dcp1/2
• 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

5
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

6
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 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
7
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

8
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
• 2024 nucleotides upstream EJ
• Also role in regulating normal gene expression

9
Non-sense mediated decay (NMD) - II

• 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
• 2024 nucleotides upstream of every
• Also role in regulating normal gene expression

Most deadenylation-independent decapping in P
bodies
10
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)

11
No-go decay

• Detecting stalled ribosomes
• Endonucleolytically cleaving the mRNA
• Dom34-Hbs1 needed for initial cleavage
• decayed by the exosome and Xrn1

12
Signals that control mRNA decay

• AU-rich elements (ARE)
• Stability element
• 3UTR 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
• mRNAunstablefacilitate rapid changes if mRNP is
  changed
• P38 MAPK, ERK, JNK, Wnt/ß-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
• aCP1 and aCP2 bind
• Protecting poly(A) tail from deadenylation

13
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
• CCR4NOT 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

14
Post-transcriptional downregulation by non-coding
RNAs
15
Key differences mRNA decay

• Bacterial decay
• Transient addition of poly(A) tailscrucial for
  exonucleolytic degradation of stem-loop
  structures
• Pyrophosphohydrolase conversion of 5 terminal
  triphosphate to monophosphate
• ? more susceptible to 5 monophosphate dependent
  RNaseendonuclease 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
  RNaseexonuclease XRN1
• Quality control PTC, recognise abnormal 3 UTR
• Non-stop decay Ski7
• No-go decay endonuclease

16
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 eventsdominant
  (deadenylation, decapping, exosomes)
• Endonucleasesmuch 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

17
The End