Stability of mRNA

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Stability of mRNA

• Structure features of eukaryotic mRNA
  untranslated regions (UTR)
• Regulation of mRNA stability in mammalian cells
• Bioinformatic analysis of UTR functional
  characterization

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Significance of mRNA Stability

• Controlling the rate at which the mRNA decays
  can regulate the levels of cellular messenger RNA
  transcripts. Because decay rates affect the
  expression of specific genes, they provide a cell
  with flexibility in effecting rapid change.

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• mRNA abundance is determined by balancing
  transcription and RNA decay. mRNA stability can
  be rapidly modulated to alter the expression of
  specific genes thereby providing flexibility in
  affecting changes in patterns of protein
  synthesis.

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• In eukaryotes the 3 poly(A) tail confers
  stability.
• In bacteria a hairpin structure in mRNA with
• r-independent terminator confers stability.

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• Moreover, many clinically relevant
  mRNAs--including several encoding cytokines,
  growth factors and proto-oncogenes--are regulated
  by differential RNA stability.

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• Most mammalian mRNAs are polyadenylated!

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mRNA Decay and Translation

• The intimate relationship between mRNA
  decay and translation is further indicated by the
  ability of translation-initiation factors (eIF)
  and proteins (PAB) that bind the poly(A) tail to
  protect the mRNA from degradation.
• Moreover, evidence shows that inhibiting
  translation elongation promotes mRNA
  stabilization.

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Translation initiation complex
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• What is the rate-limiting step in mRNA
  degradation?
• An evolutionarily conserved
  mRNA-degradation pathway is initiated by the
  removal of the 3- poly(A) tail. This disrupts
  the translation initiation complex and provides
  degradative enzymes with access to the 5 cap and
  remaining RNA body.

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• What are the sequence elements and factors
  that control the half-lives of mRNAs?

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General Structure of a Eukaryotic mRNA

• illustrating some post-transcriptional
  regulatory elements for gene expression and their
  activity. 5UTR mediated regulation may involve
  the 7-methyl-guanine (cap) hairpin-like
  secondary structure RNA-protein interactions
  upstream open reading frames (uORFs) internal
  ribosome entry sites (IRES). 3UTR mediated
  regulation may involve antisense RNA
  interactions RNA-protein interactions, involving
  also multiprotein complexes cytoplasmic
  polyadenylation elements (CPE) poly(A) tail and
  variation of its size.

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Elements in the 5 UTR
7-methylguanosine cap
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• Caps provide at least four functions
• Protection of mRNA from degradation (see
  example)
• Enhancement of mRNAs translatability
• Transport of mRNA out of nucleus
• Proper splicing of pre-mRNA

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• Furuichi colleagues, 1977
• Labeled reovirus RNAs w/ capped (green),
• blocked (blue) or decapped (red) 5-end
• a) glycerol gradient centrifugation (ggc)
• b) incubation in X. oocyte, 8 h gt ggc
• c) decapped deblocked RNAs
• (b-elimination yielding pppGm pppG)
• incubation in X. oocyte, 8 h gt ggc
• d) as (b) but incubation in wheat germ extract

w/o w/
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Elements in the 3 UTR

• AU-rich element (ARE) and proteins that bind AREs
• Iron-responsive element (IRE) and iron regulatory
  protein (IRP)
• Cell cycle-regulated histone mRNA stem-loop
  determinant (SL/SLBP)
• Cytoplasmic polyadenylation element (CPE)

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Transferrin receptor mRNAion-responsive element
IRP
PNAS 93 8179-8182, 1996
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Histone mRNA stem loop determinant SLBP

• Gene 239 1-14, 1999

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assembly
CPSF blue CstF brown CF I II grey
cleavage
stimulated by pol II CTD CstF CFs leaves PAP
(orange) enters
Cytoplasmic polyadenylation elements (CPE)
binding proteins
oligo(A) synthesis aided by CPSF
poly(A) synthesis aided by PAB II (yellow)
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Darnell colleagues, 1973 Poly(A) nuclear form
210 nt, cytoplasmic form 190 nt gt poly(A)
undergoes considerable shortening in the cytoplasm
Nuclear cytoplasmic
5S rRNA
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Cytoplasmic polyadenylation of maternal
mRNAs The best studied cases are those that
occur during oocyte maturation Maturation-specifi
c polyadenylation of Xenopus maternal mRNAs in
the cytoplasm depends on two sequence motifs The
AAUAAA motif an upstream motif with UUUUUAU or
a closely related sequence
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Wickens colleagues, 1989 inject labeled RNAs
into X. oocyte cytoplasm gt stimulate
maturation w/ progesterone gt isolate RNAs
electrophoresis
Lack UUUUUAU

• maturation-specific (i.e., D7) RNAs, containing
  UUUUUAU,
• was polyadenylated

P pregesterone A separation by oligo(dT)
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• AAUAAA is also required for cytoplasmic
  polyadenylation
• this motif is required for both nuclear
  cytoplasmic polyadenylation

SV40 RNA
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Poly(A) is not just shortened in the cytoplasm
it turns over! RNases tear it down, and poly(A)
polymerase builds it back up When the poly(A)
is gone, the mRNA is slated for destruction
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Stability of mRNA

• Structure features of eukaryotic mRNA
  untranslated regions (UTR)
• Regulation of mRNA stability in mammalian cells
• Bioinformatic analysis of UTR functional
  characterization

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mRNA Decay Pathways in Mammalian Cells

• Deadenylation-dependent pathways
• Deadenylation-independent pathways
• 1. endoribonucleolytic decay
• 2. nonsense-mediated decay (NMD)
• RNAi-dependent pathway

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Deadenylation-dependent mRNA Decay

• When mRNA processing is complete, the mRNA
  bears a 5' cap structure and 3' poly(A) tail that
  protect the message from exonucleolytic decay.
  The first step in the decay of most wild-type
  mRNAs is shortening of the poly(A) tail by a
  deadenylase (blue). Once poly(A) shortening is
  complete, the 5' 7-methylguanosine cap is rapidly
  removed and the rest of the mRNA is attacked by
  5' and 3' exonucleases (green and pink,
  respectively).

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• The turnover of mRNAs is also regulated by
  cis-acting elements that either promote or
  inhibit their decay. The most prevalent is the
  AU-rich element (ARE), found in the
  3-untranslated region (3 UTR) of mRNAs encoding
  many important growth control proteins.

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The deadenylase as an inhibitor of translation
initiation and decapping

• During translation, the mRNA is thought to be
  circularized by its interaction with the
  translation-initiation factors eIF4E (4E), eIF4G
  (4G) and the poly(A)-binding protein (PABP). This
  conformation protects the 3' and 5' ends of the
  mRNA from attack by the deadenylase and decapping
  enzymes. The deadenylase can somehow invade this
  closed loop and interact with the cap while
  simultaneously removing the poly(A) tail. The
  interaction of poly(A) ribonuclease (PARN) with
  the cap perpetuates the closed loop and thereby
  blocks both translation initiation and decapping.
  

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When poly(A) shortening is complete, PARN
dissociates, allowing the decapping enzyme to
hydrolyse the 5' cap of the message. Meaning
that hydrolysis the 5' cap of mRNA depends on
the completion of poly(A) shortening by PARN.
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• In addition,
• ARE-binding proteins affect mRNA stability,
  translation and subcellular localization.
• Other elements found in the 5 UTR and coding
  regions also modulate transcript stability.

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• Signalling pathways also affect
• mRNA stability
• Several signaling pathways are implicated in
  triggering changes in stability of specific
  mRNAs.
• e.g., interleukin-2 mRNA, which is stabilized
  by the c-Jun amino-terminal kinase (JNK)
  signaling pathway through JNK-responsive elements
  in its 5 UTR.

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AU-rich element The most prevalent cis-acting
element Class I AREs contain 1 to 3 scattered
copies of the pentanucleotide AUUUA embedded
within a U-rich region, and are found in the
c-Fos and c-Myc mRNAs. Class II AREs contain
multiple overlapping copies of the AUUUA motif,
and are found in cytokine mRNAs. Class III
AREs, such as the one in c-Jun mRNA, lack the
hallmark AUUUA pentanucleotide but present
U-rich sequences.
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Model for how the ARE mediates stability and
instability

• Interaction of the ARE with a destabilizing
  factor, such as AUF1 to c-myc mRNA, might
  promote rapid deadenylation by reducing the
  affinity of the poly(A) binding protein (PABP)
  for the poly(A) tail.
• Conversely, stabilizing factors, such as HuR
  to VEGF mRNA in response to hypoxia, might
  enhance binding of the PABP to the poly(A) tail,
  thus blocking deadenylation.

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Some other ARE-BPs have been proven to possess
destabilizing activity on ARE-RNAs TTP, BRF1,
KSRP AUF1 has a dual role in ARE-mediated mRNA
decay, functioning either as a destabilizing or
a stabilizing factor depending on the cell type.
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mRNA Decay Pathways in Mammalian Cells

• Deadenylation-dependent pathways
• Deadenylation-independent pathways
• 1. endoribonucleolytic decay
• 2. nonsense-mediated decay (NMD)
• RNAi-dependent pathway

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Endoribonucleolytic Decay

• There are a few messenger RNAs that degrade
  by a minor pathway known as endoribonucleolytic
  decay. Endoribonucleases recognize specific
  sequence elements within the transcript and
  cleave the mRNA internally. The cleavage event
  generates free 3' and 5' ends that are easily
  accessible to exonucleases and the products of
  the cleavage reaction are therefore rapidly
  degraded.
• In contrast, stabilizer protein may block the
  binding of endoribonuclease.

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• Interestingly, several mRNAs that are
  degraded by endoribonucleolytic decay also
  interact with stabilizer proteins that block
  access of the endoribonuclease to its cleavage
  site.
• e.g., an endonuclease from Xenopus laevis
  hepatocytes, PMR1, can cleave the vitellogenin
  mRNA but its action is prevented by binding of
  the vigilin protein to a site that overlaps the
  PMR1 cleavage site.
• Similarly, the a-globin mRNA is cleaved at a
  site in its 3' UTR by an erythroid-enriched
  endonuclease. In this case, cleavage is inhibited
  by binding of the a-CP complex of proteins to an
  overlapping sequence.

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• A strong link between translation and RNA
  turnover is also shown by nonsense-mediated decay
  (NMD), which ensures that mRNAs containing
  premature stop codons are degraded. (evidence
  from yeast studies)

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NMD Pathways

• NMD prevents the accumulation of aberrant
  transcripts and truncated proteins by ensuring
  rapid decay of the mRNAs.
• Targets transcripts that harbor nonsense
  codons, unspliced introns or extended 3UTRs.

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• In the yeast S. cerevasiae, NMD involves
  deadenylation-independent decapping followed by
  5-3 degradation of mRNA.
• In mammalian cells, the sequence of degradation
  steps involved in NMD is unknown.

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NMD of alternatively spliced mRNA
(Bioinformatics 19 118-121, 2003)
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Translation OK
Remaining EJCs trigger NMD
(Bioinformatics 19 118-121, 2003)
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Regulated mRNA Stability

• Regulation in response to developmental/differenti
  ation cues
• Regulation in response to hormonal regulation
• Regulation in response to diurnal variation
• Regulation in response to stress

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Deregulated mRNA Stability

• Deregulation in carcinoma
• Deregulation in Alzheimers disease

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• How is RNA decay regulated in bacteria?
• The stability of mRNA in the cytosol of eukarya
  is increased by the addition of a 3 poly(A)
  extension.
• By contrast, this process mediates rapid RNA
  decay in prokarya.

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How is mRNA decay regulated in mitochondria?

• Their monophyletic, a-proteobacterial origin
  predicts that polyadenylation will induce rapid
  decay by nucleases and associated factors that
  are similar to their bacterial ancestors.
• Is it true?

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The role of polyadenylation in different
mitochondrial (mt) systems.
Trends in Genet. 20 260-267, 2004
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mRNA Decay Pathways in Mammalian Cells

• Deadenylation-dependent pathways
• Deadenylation-independent pathways
• 1. endoribonucleolytic decay
• 2. nonsense-mediated decay (NMD)
• RNAi-dependent pathway

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2006 Nobel Prize in Physiology/Medicine "for
their discovery of RNA interference gene
silencing by double-stranded RNA"
Craig C. Mello U. Mass.
Andrew G. Fire Stanford
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2006 Nobel Prize in P/M RNA interference (RNAi)
Original paper Nature, 1998
Breakthrough of the year Science, 2002
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What is RNA interference? RNA Interference
(RNAi) was first identified as a
post-transcriptional response to exogenous
double-stranded RNA (dsRNA) introduced into the
nematode worm, C. elegans, and is largely
conserved from fungi to plants to mammals. The
pathway is triggered when long dsRNA encounters
the RNaseIII enzyme Dicer, a cytoplasmic enzyme
that cleaves the dsRNA to produce short,
interfering RNAs (siRNAs). One strand of the
siRNA is incorporated into the effector complex
of RNAi, the RNA-induced Silencing Complex
(RISC). The short RNA guides RISC to target mRNA
and catalyzes an endonucleolytic cleavage,
resulting in a post-transcriptional silencing of
gene expression.
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(by P. Sharp)
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Cullen, BR Virus Res., 2004
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Stability of mRNA

• Structure features of eukaryotic mRNA
  untranslated regions (UTR)
• Regulation of mRNA stability in mammalian cells
• Bioinformatic analysis of UTR functional
  characterization

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Bioinformatic Analysis

• UTRdb collecting UTR sequences
• UTRsite collecting UTR specific regulatory
  signals

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UTRdb

• UTRdb is a non redundant database of 5 and
  3UTR sequences generated by a computer program
  through the parsing of EMBL/GenBank database
  entries.
• A summary description (release 14.0, Jan 2001)
  presently contains gt 120,000 entries accounting
  for gt 40,000,000 nucleotides.

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UTRsite

• UTRsite is a specialized database that
  collects UTR specific regulatory elements. Each
  UTRsite entry includes a summary description of
  the biological role of the corresponding element,
  the relevant pattern consensus structure and the
  related bibiography.

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