GPLS 701 Advanced Molecular Biology Messenger RNA Turnover

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GPLS 701 Advanced Molecular BiologyMessenger
RNA Turnover

• Gerald Wilson, Ph.D.
• Department of Biochemistry and Molecular Biology
• BRF Room 239
• gwils001_at_umaryland.edu

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Todays Menu

• Principles of cellular mRNA decay
• Mechanisms
• Methodology
• Signals and Factors
• AU-rich element-directed mRNA decay
• microRNAs and small interfering RNAs
• Synthesis and Processing
• Functions

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Gene Expression in Eukaryotes
(Orphanides and Reinberg (2002) Cell 108, 439-451)
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Contributions of mRNA turnover to control of gene
expression - I

• The translational potential of an mRNA is
  dependent upon its cytoplasmic concentration.
• Steady-state cytoplasmic mRNA concentrations are
  combined functions of the rates of
• Synthesis
• -transcription
• -pre-mRNA splicing
• -nucleocytoplasmic transport
• Cytoplasmic turnover

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Contributions of mRNA turnover to control of gene
expression - II

• Experimentally, changes in the concentration of a
  cytoplasmic mRNA can be modeled by
• These properties of mRNA accumulation kinetics
  allow mRNA turnover rates to influence gene
  expression by two principal means
• Modulation of decay rate in response to some
  stimulus (eg hormonal, developmental,
  environmental, etc.)
• Kinetics of steady-state approach following
  changes in synthetic rate

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Example 1 An mRNA with a 15 minute half-life is
stabilized 10-fold under constant transcription
rate. After 3 hours, the decay rate returns to
give a 15 minute half-life.NB t1/2 ln2/kdecay
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Example 2 The synthetic rates of mRNAs with 15
minute (solid line) or 5 hour (dashed line)
half-lives are increased 10-fold. After 3 hours,
syntheses return to their original rates.
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A point to pondermRNA synthesis is
energetically expensive. What situations would
prompt cells to deliberately make mRNAs, only to
have them rapidly destroyed?
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Determination of cellular RNA decay rates - I

• 32P-UTP pulse-chase
• useful only for abundant, long-lived RNAs (slow
  cellular uptake of UTP, high background due to
  bulk RNA)
• Approach to steady-state kinetics
• following change in transcription rate
• requires knowledge of kinetics of RNA synthesis
• difficult to resolve in most cases

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Determination of cellular RNA decay rates - II

• Global inhibition of RNA pol II transcription
• cells treated with actinomycin D or DRB
• specific mRNA quantitifed as a function of time
  following transcriptional arrest (Northern, RPA,
  RT-PCR)
• RNA decay observed within a dying cell population
• Inducible promoters
• RNA of interest expressed from inducible promoter
  like fos (serum-responsive) or Tet-based systems
• cannot use to measure decay kinetics of
  endogenous mRNAs

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Actinomycin D time course assay
TPA
TPA
control
control
(Wilson et al, (2003) J. Biol. Chem. 278,
33029-33038)
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Calculation of mRNA decay rates
For single-phase exponential decay, RNAt
RNA0e-kt t1/2 ln2/k
TPA
TPA
control
control
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Schematic of a mature eukaryotic mRNA
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Eukaryotic mRNA Decay Pathways
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Roles of Nucleases

• Since mRNA turnover is a catabolic process,
  sooner or later nuclease(s) must be recruited to
  degrade the RNA substrate.
• Candidates
• Poly(A)-specific ribonuclease (PARN)
• First identified in Xenopus cytoplasm
• Shows high specificity for poly(A) sequences
• Probably functions to regulate translation in the
  oocyte
• Exosome
• Protein complex containing 11-14 3-5
  exoribonucleases
• Demonstrated to rapidly degrade poly(A)- RNA
  substrates
• Proteasome?
• Inhibition of the multi-subunit protease complex
  stabilizes some mRNAs
• AU-rich RNA fragments co-purify with 26S
  proteasome
• Other soluble nucleases
• homologs of yeast Caf1, Pop2, etc?

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How are stable versus unstable mRNAs
discriminated?Cis-acting elements

• 3-untranslated region
• AU-rich elements (AREs)
• Iron-responsive elements (IREs)
• Many other poorly defined sequences
• Coding region
• c-myc coding region determinant
• 5-untranslated region
• Translational regulators
• JNK response element (JRE)

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Cis-acting mRNA stability determinants are
identified using chimeric mRNAs
(Fialcowitz et al. (2005) J. Biol. Chem. 280,
22406)
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Cis-acting elements mediate their effects by
interaction with trans-acting factors

• For many cis-acting determinants of RNA
  stability, sequence-specific RNA-binding proteins
  have been identified
• The RNA-binding activity of these factors may
  correlate with RNA stabilization,
  destabilization, or may be independent of changes
  in the mRNA turnover rate
• Individual cis-acting sequences may be targeted
  by multiple RNA-binding proteins

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Detection/Identification of trans-acting factors
- I
Gel mobility shift assay (GMSA)/ antibody
supershift
UV cross-linking
(Wilson and Brewer (1999) Methods, 17, 74)
(Wilson et al, (2003) J. Biol. Chem. 278, 33029)
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Detection/Identification of trans-acting factors
- II

• RNA affinity chromatography (biotin pulldown
  assay)

Identify sequence of interest in target mRNA

PCR product (downstream of bacteriophage promoter)
T7
-detect candidate RNA-binding proteins by
Western blot -identify unknown RNA-binding
proteins by mass spectrometry
In vitro transcription (incorp Biotin-CTP)
B
B
B
B
B
B
Incubate with cellular extract
B
B
B
B
B
B
Bind to streptavidin-coated beads
B
B
B
B
B
B
Streptavidin- -coated Beads
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Specific ExampleAU-rich Elements
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AU-rich elements (AREs)

• potent cis-acting determinants of rapid mRNA
  turnover in mammalian cells
• are present in the 3'-UTRs of many labile mRNAs,
  including several encoding inflammatory
  mediators, cytokines, oncoproteins, and G
  protein-coupled receptors
• diverse in size (40-150 nt) and sequence, but
  generally consist of one or more AUUUA pentamers
  contained within or near a U-rich tract
• mRNA turnover mediated by AREs is usually
  characterized by rapid 3' to 5' shortening of the
  poly(A) tail followed by decay of the mRNA body

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Representative AREs from human mRNAs
GM-CSF 5- UAAUAUUUAUAUAUUUAUAUUUUUAAAAUAUUUAUUUAU
UUAUUUAUUUAA -3 IL-3 5- AUUUAUUUAUGUAUUUAUGUAUU
UAUUUAUUUA -3 TNFa 5- AUUAUUUAUUAUUUAUUUAUUAUUU
AUUUAUUUA -3 c-fos 5- UUUUAUUGUGUUUUUAAUUUAUUUA
UUAAGAUGGAUUCUCAGAUAUUUAUAUUUUU
AUUUUAUUUUUUUU -3 c-myc 5- AUAAAAGAACUUUUUUAUGC
UUACCAUCUUUUUUUUUUCUUUAACAGAUUUGUAUU
UAAGAAUUGUUUUUAAAAAAUUUUAAGAUUUACACAAUGUUUCUCUGUAA
AUAUUG CCAUUAAAUGUAAAUAACUUUAAU
-3 c-jun 5- UUUCGUUAACUGUGUAUGUACAUAUAUAUAUUUUU
UAAUUUGAUUAAAGCUGAUUA CUGUGAAUAAACAGCUUCAUGCC
UUUGUAAGUUAUUUCUUGUUUGUUUGUUUGGG
UAUCCUGCCCAGUGUUGUUUGUAAAUAAGAGAUUUGGAGCA -3
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Selected cellular ARE-binding factors
Function
Expression site
RNA-binding motif
Factor
mRNA destabilization
ubiquitous
RRM
AUF1
unknown
T cells
unknown
AU-A, -B, -C
unknown
ubiquitous
RRM
hnRNPs A0, A1, C
mRNA stabilization
ubiquitous
RRM
HuR
mRNA stabilization
neuronal
RRM
HuC, HuD
translational silencing
ubiquitous
RRM
TIA-1/TIAR
mRNA destabilization
ubiquitous?
CCCH Zn fingers
TTP
mRNA destabilization
ubiquitous
KH domains
KSRP
unknown
ubiquitous
Rossman fold
GAPDH
unknown
ubiquitous (hs)
unknown
Hsp70
miR16
base pairing
ubiquitous?
mRNA destabilization
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AUF1 an ARE-targeted mRNA destabilizing factor

• first identified as a component of a cytoplasmic
  complex which accelerated ARE-directed mRNA
  turnover in a cell-free mRNA decay system
• affinity of AUF1 for an ARE correlates with the
  mRNA-destabilizing activity of the ARE

(adapted from DeMaria and Brewer (1996) J. Biol.
Chem. 271, 12179)
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Alternative pre-mRNA splicing generates four AUF1
isoforms
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How does AUF1 function?

• AUF1 oligomerizes on ARE sequences by sequential
  binding of protein dimers
• AUF1 induces local condensation of RNA structure
• Biophysical analyses suggest that surface area is
  maximized in the AUF1ARE complex

(Wilson et al. (2003) J. Biol. Chem. 278, 33039)
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AUF1 associates with other cellular factors

• AUF1 first identified as a component of a
  multi-subunit complex.
• (Brewer (1991) Mol. Cell. Biol. 11, 2460)
• Several proteins and phosphoproteins
  co-immunoprecipitate with AUF1. (Zhang et al
  (1993) Mol. Cell. Biol. 13, 7652)
• Some factors interacting with cytoplasmic AUF1
  have been identified by tandem co-i.p./Western
  analyses.
• (Laroia et al (1999) Science 284, 499 - right)

35S-cell label/ AUF1 i.p.
Tandem i.p./ Westerns
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HuR an ARE-targeted mRNA stabilizing factor

• HuR is a 36 kDa member of the Hu protein family,
  which shows homology to the Drosophila Elav
  (Embryonic lethal abnormal vision) protein.
• In mammals, the family includes the ubiquitously
  expressed HuR, and the neuronal-specific Hel-N1
  and HuD.
• All Hu proteins contain 3 RRMs in a
  characteristic arrangement (right)
• Overexpression studies indicate that Hu proteins
  stabilize ARE-containing mRNAs.

(GMSA)
(Ma et al. (1996) J. Biol. Chem. 271, 8144)
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Putting it all together A model for mRNA decay
targeted by AREs
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More Points to Ponder (and good exam questions)

• Many different proteins bind common ARE targets.
  Why might evolution have devised such a system?
• Some AREs, especially from proto-oncogene mRNAs,
  are quite large (gt150 nt), yet ARE-binding
  proteins only require (on average) 9-20 nt of RNA
  for sequence-specific binding. Why are the large
  ARE sequences necessary?
• When describing the activity of a trans-acting,
  RNA-binding factor, a common (but dangerous)
  reviewers request is an overexpression
  experiment. Why might overexpression of a factor
  that promotes mRNA decay actually result in mRNA
  stabilization?

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microRNAs metazoan trans-regulators of gene
expression

• microRNAs (miRNAs) are derived from processed
  endogenously expressed precursor RNAs encoded by
  genomic loci distinct from other known genes.
• small interfering RNAs (siRNAs) can be derived
  from exogenous sources (ie viruses or synthetic
  sources) or by-products of endogenous RNAs
  (introns, transposons, etc.) although they may be
  processed within the cell

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miRNA precursors

• miRNA precursors have extended intramolecular
  base pair potential
• RNA bulges and kinks are common

(Bartel (2004) Cell 116, 281-297)
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Processing miRNAs and siRNAs
(Tomari and Zamore (2005) Genes Dev. 19, 517-529)
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Strand selectivity of miRNAs and siRNAs is based
on local duplex stability - I
(Tomari and Zamore (2005) Genes Dev. 19, 517-529)
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Strand selectivity of miRNAs and siRNAs is based
on local duplex stability - II
(Tomari and Zamore (2005) Genes Dev. 19, 517-529)
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Consequences of miRNA and siRNA targeting to
complementary RNA (or DNA) substrates - I
(adapted from Bartel (2004) Cell 116, 281-297)
Extensive complementarity between miRNA/siRNA and
mRNA coding regions or 3UTRs induce mRNA
cleavage and turnover
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Consequences of miRNA and siRNA targeting to
complementary RNA (or DNA) substrates - II
(adapted from Bartel (2004) Cell 116, 281-297)
Short regions of complementarity between
miRNA/siRNA and mRNA 3UTRs can inhibit
translation
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Consequences of miRNA and siRNA targeting to
complementary RNA (or DNA) substrates - III
(adapted from Bartel (2004) Cell 116, 281-297)
Complementarity between miRNA/siRNA and DNA can
inhibit transcription