Concerted action of polyA nucleases and decapping enzyme in mammalian mRNA turnover

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Concerted action of poly(A) nucleases and
decapping enzyme in mammalian mRNA turnover

• Akio Yamashita1, 2, Tsung-Cheng Chang1, 2, Yukiko
  Yamashita1, Wenmiao Zhu1, Zhenping Zhong1,
  Chyi-Ying A Chen1  Ann-Bin Shyu

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(No Transcript)
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Goals of the paper

• Do these deadenylases exist and act as they do in
  yeast turnover of mRNA
• How do they affect the kinetics of normal and NMD
  mRNA turnover
• What effect does decapping have on these
  processes

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Analogs to yeast cytoplasmic deadenylation
factors are found in mammalian cells
Figure 1a Endogenous protein localization in
NIH3T3 cells based on western blot analysis of
sub cellular distributions on the
(Left) Transfected HA-tagged CCR4a and CCR4b
distribution analysis by western blot (Top
Right) (Bottom Right) mRNA knockdown of
endogenous CCR4a and b Figure 2a Confocal
microscopy of tagged proteins in live cells
further confirm the western blot data Cytoplasm
(predominantly) CCR4, CAF, PAN2 and
PAN3 Nucleus PARN
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These proteins form similar complexes to their
yeast analogs
Figure 1b Protein interactions were detected by
co-IP in Figure 1b using HA tagged
proteins. CCR4a/b-CAF1a PAN2-PAN3
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Deadenylation proteins shuttle between the
nucleus and cytoplasm
Leptomycin B inhibits exportin-1 Figure 2b
Nuclear localization of deadenylation complexes
when nuclear export is blocked
Figure 2c PARN is found in the nucleus of
cyclohexamide fused HeLa cells
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Biphasic deadenylation of mRNAs
Figure 3 Deadenlyation occurs in 2 distinct
steps shortening of the poly a tail
(synchronous) further shortening of the tail and
degradation of the mRNA body (asynchronous) Phase
2 appears sooner in PTC containing mRNAs
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Pan2 and Ccr4 act sequentially in biphasic
deadenylation
(Figure 4) CCR2 mediates phase 2 degradation and
PAN2 mediates phase 1 of deadenylation of
b-globin mRNA as determined by overexpression.
PARN does not affect the course of this mRNA
despite higher expression levels Inactive
variants of the proteins are transfected to
verify that it is not an artifact of
overexpression
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Because PARN does something
Figure 4 In vitro degradation of Poly(A)
substrate by WT is greater than that of MT
however it is not a complete loss of function
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Reduction of phase 2 activity by CCR4 RNAi
Figure 5 siRNA knockdown of CCR4a/b prevents the
onset of asynchronous degradation PARN has
supposedly very little effect on the kinetics of
decay
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Nucleases in NMD
Figure 3 Control experiment for normal NIH3T3
cells
Figure 6 Over expression of the above labeled
constructs in the NIH3T3 cells compared to
control results from figure 3 NMD Ccr4 and Pan2
play roles identical to that of normal mRNA
processing in NMD
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Nucleases in NMD cont.
Figure 6 NMD under condition of reduced CCR4 and
PARN expression by siRNA knockdown As before,
knockdown of CCR4a/b stabilizes the mRNA
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Deadenylation of mRNA can occur independently of
decapping in eukaryotes
Accumulation of intermediates can be detected in
the absence of decapping Dcp2 indicating that
Dcp2 likely acts after the first phase has
completed This also seems to indicate that some
mRNA must be decapped prior to deadenylation by
Ccr4
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Deadenylation can occur independently of
decapping in eukaryotes cont.
Moderate stabilization of PTC mRNA may indicate
mammals depend on decapping mediated decay
compared to yeast, which is almost entirely
independent of decapping.
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Proposed model of mRNA clearance
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Conclusions

• PAN2 is primarily involved with the initial
  degradation of the poly(A) tail
• CCR4 is responsible for asynchronous degradation
  of the mRNA
• Both are functionally linked and involved
  similarly in NMD

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Questions

• What are the factors involved in the interaction
  between deadenylation and decapping
• How do these factors respond to different mRNA
  substrates
• How does altering shuttling activity of the
  protein affect function in deadenylation

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Decapping and Decay of Messenger RNA Occur in
Cytoplasmic Processing Bodies

• Ujwal Sheth and Roy Parker

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Claims

• mRNA decapping and 5-3 degradation occurs in
  discrete cytoplasmic foci (P bodies)
• Proteins that activate or catalyze decapping are
  found in P bodies
• Inhibiting mRNA turnover prior to decapping leads
  to reduced P bodies however, inhibiting turnover
  at or after decapping increases the number of P
  bodies
• Degradation mediates are localized to P bodies

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Many proteins involved in mRNA turnover localize
to punctate structures
Figure 1 (Left) Dcp1, Dcp2, Pat1, Lsm1,Ccr4
(discrete, transient), Dhh1 and Xrn1 with
fluorescent epitopes have a punctate distribution
in cells Figure 2 Mutations of the mentioned
proteins and a look at distribution with P body
marker Dhh1 GFP tagged Dcp1 and Xrn1 mutants show
increased P body size and number while Ccr4
(deadenylation) and Pat1(Decapping activator)
mutants show marked reduction
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Degradation mediates are localized to P bodies
Figure 3a Poly G inserted in MFA2to trap the
decay intermediate therefore, no accumulation
when not present Figure 3b Co-localization of
these intermediates with Lsm1 indicating that
mRNAs in the process of decay are associated with
P bodies Figure 3c Reproducible results using
the PGK reporter mRNA
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Questions

• What factors are required for the localization of
  mRNA in P bodies? Pat1 vs Lsm1
• Are some of these factors only transiently
  associated with P bodies and what would that time
  course be?
• Are P bodies associated with NMD