mRNA Decay
1
mRNA
eIF4G
Pab1p
eIF4E
m7Gppp
UAA
AUG
AAAAAAAAA
eIF3
m7Gppp
A
A
A
A
A
A
A
AUG
UAA
2
Regulatory elements of mRNA
mRNAs contain several regulatory elements recognized by specific
regulatory factors: proteins and RNAs, which allow fine-tuned
regulation of gene expression by controlling its translation, degradation
and localization.
Pathways by which eukaryotic mRNAs
are degraded
4
1
3
2
ARE mRNAs
9E3
transferrin receptor
c-myc
Vitellogenin
β-globin
RNAi
4
Cytoplasmic mRNA degradation
Stabilisation
Cap
binding
complex
PAB1
PAB1PAB1
m7GpppN
2
CCR4
POP2
NOT
AAAAAAAAAAAA
LSM 1-7
Decapping
1
PAB1
DCP1
DCP2
m7GpppN
AA
CCR4
POP2
NOT
Deadenylation
XRN1
DCP1
DCP2
XRN1
( 5 ’->3 ’)
pN
A
Exo
3’>5 ’
Exosome
5
Different mRNA decays operate in yeast
and mammals
Ccr4;Not;Pop2
DcpS
Dcp1;Dcp2
Exosome
Lsm 1-7
Xrn1
yeast
5’->3’
degradation
DcpS
Exosome
Mammals
3’->5’
degradation
6
Eukaryotic mRNA decay
Eukaryotic mRNA deadenylases
1.
2.
3.
*
*Only in metazoa
Yeast mRNA Deadenylase
In yeast, a poly(A) nuclease, referred to as PAN, has been identified
biochemically and shown to be an enzyme consisting of the Pan2 and
Pan3 proteins
The Pan2p subunit is likely the catalytic subunit since this protein is a
member of the RNaseD family of 3′ to 5′ exonucleases
However, pan2Δ and pan3Δ yeast strains show minimal effects on
deadenylation in vivo (required for initial shorthening-20nt)
Ccr4p and its associated factor, Pop2/Caf1p, have nuclease domains
(Mg2+ dep. endonuclease and RNaseD family)
Ccr4Δ and Pop2/Caf1Δ yeast strains show defects on deadenylation in
vivo
Ccr4Δ/pan2Δ strain is completely blocked for deadenylation
How to study mRNA decay in yeast
• Transcription must be off
(GAL or Tet-OFF
promoter)
• The PolyG cassette (pG)
blocks exonuclease
activity
Ccr4p and Pop2/Caf1p Are Required for
Normal Rates of mRNA Turnover
Measurement of the Decay Rate of the MFA2pG mRNA:
Glucose
Pop2/
Pab1p inhibits Ccr4p deadenylation activity
Analysis of deadenylation activity in Flag-Ccr4p purified
fractions with addition of increasing amounts of purified
Pab1p
The initial phase of poly(A) trimming
is carried out by PAN2, which
interacts directly with and is
stimulated by PABP and is additionally
regulated by its binding partner
PAN3. Subsequently, bulk mRNA
deadenylation is the responsibility of
the conserved CCR4–NOT complex,
which contains the Ccr4 and Pop2
(Caf1) 3′–5′ exonucleases and the
scaffold protein Not1. CCR4, the
major deadenylase of this complex, is
inhibited rather than activated by
PABP. As it is also required for
translation initiation, it seems that
PABP plays a central part in
coordinating poly(A) tail length,
translation and deadenylation.
Deadenylation complex
Deadenylation leads to the release of PABPs from the mRNA 3’ –end which allows
for a direct attack by 3’ -5’ exoribonucleases.
A major eukaryotic exoribonuclease degrading transcripts from 3’ -end is
the RNA exosome complex
In yeast, exosome-mediated 3’ -5’ decay pathway, although functional, does not
play a major role in the control of mRNA stability, and decapping-dependent 5’ -3’
pathway prevails.
yeast
Mammals
Exosome
Exosome's cellular functions and cofactors
The exosome is a large protein complex containing multiple 3′→5′ exonucleases
that also functions in a variety of nuclear RNA processing reactions
Cofactors for the exosome complexes
Cytoplasmic exosome
Ski2p,Ski3p, Ski8p
Ski2p - DEAD box RNA helicase ATP
dependent. Remove structures and /or
protein to allow exosome digestion
Ski7p - putative GTPase homologous
to translation factors (e.g. EF-Tu and
EF-G)
Barrel like
Function:
•mRNA degradation
In human there are three proteins of the DIS3 family: DIS3, DIS3L and DIS3L2.
Of these, DIS3L and DIS3L2 are cytoplasmic
mechanisms of exosome recruitment to
different mRNA substrates
17
Regulatory elements of mRNA
mRNAs contain several regulatory elements recognized by specific
regulatory factors: proteins and RNAs, which allow fine-tuned
regulation of gene expression.
ARE-mediated decay (AMD)
•Several sequence elements within transcripts have been linked to the
control of mRNA turnover. AU-rich elements (ARE) in 3'-UTR
characterize most short-lived transcripts
•Destabilizing RNA-binding proteins have been shown to direct AREcontaining mRNA to the exosome
•RNA can be stabilized by proteins that compete for ARE binding and
direct transcripts to the polysome for translation
•AREs are found in mRNAs involved in cellular responses to
environmental and/or metabolic changes: cyclins, cytokines (GM-CSF,
IL-3 etc.), oncogenes (c-myc, c-fos etc.)………
ARE binding proteins
(tristetrapolin)
These proteins have been identified in cell
ultraviolet (UV)-crosslinking and gel-shift assays
extracts
by
AREs drive mRNA deadenylation
The binding of the ARE-BPs TTP and KSRP on AREs induces rapid
mRNA deadenylation by the recruitment of the deadenylases
CCR4–NOT complex and PARN, respectively. The binding of HuR
stabilizes the transcript by inhibiting exosome recruitment.
The mammalian exosome mediates the
degradation of mRNAs that contain ARE
•Capped but non-polyadenylated RNAs were prepared that lacked (GemA0) or contained the ARE (GemARE-A0) and incubated in HeLa S100
extracts (Figures B and C)
•immunodepletion of HeLa extracts with antibodies against the
exosomal protein PM-Scl75 reduced the efficiency of 3'−5'
exonucleolytic decay in these assays
Down-regulation of PARN inhibits deadenylation
step of ARE-mediated Decay (AMD)
HeLa cells were transfected with a construct expressing beta globin
(GB)-AREGMCSF under the control of a TetOFF promoter
siRNAs
(control)
ARE-mRNA accumulates in cytoplasmic exosome
granules containing PARN
exosome
mRNA-Specific Recruitment of the General
Repression and Decay Complexes
Decapping
complex
Degradation
complex
Deadenylation
complex
Degradation of NMD-targeted mRNAs
In mammals, rapid degradation of NMD targets can be achieved through at least
three different pathways
1.
SMG6
cleaves
mRNA
endonucleolytically in
the vicinity of PTCs and in human cells
the majority of NMD targets are
apparently
degraded
through
this
pathway. After SMG6-mediated cleavage,
for which SMG6 needs to interact with
UPF1, the 3’ RNA fragment is rapidly
degraded by XRN1 and the 5’ fragment
seems to be digested by the exosome.
2. The heterodimer SMG5–SMG7, which interacts with phosphorylated UPF1,
Interacts the deadenylase CNOT8 (POP2) interacts with this C-terminal of SMG7,
indicating that SMG5-SMG7 recruits the CCR4-NOT complex to NMD targets to
induce their deadenylation-dependent decapping and subsequent XRN1-mediated
degradation. Consistently, SMG7-mediated mRNA decay requires the presence of
DCP2 and XRN1 but not of SMG6.
3. UPF1 also associates with decapping complex subunits DCP1A, DCP2, and PNRC2,
indicating that direct deadenylation-independent decapping might constitute a third
route to degrade aberrantly terminating mRNAs
microRNAs induce deadenylation of mRNA targets
Translational repression
/degradation
mRNA decapping
28
Ccr4;Not;Pop2
DcpS
Dcp1;Dcp2
Lsm 1-7
Decapping plays a key
role in different mRNA
degradation pathways
DcpS
Xrn1
Dcp1;Dcp2
Dcp1;Dcp2
29
Eukaryotic decapping proteins
NUDIX domains are essential
for enzymatic activity
•The Dcp1:Dcp2 complex catalyzes the removal of
from a capped RNA of more than 25 nucleotides
m7GDP
(7-methyl-GDP)
•DcpS hydrolyzes the m7GpppN cap releasing m7GMP, from capped
oligonucleotides produced by exosome and results from complete mRNA
degradation. It is unable to decap long substrates.
DcpS exists in complex with the exosome and is believed to play an important role in ensuring that no
excess unhydrolyzed cap accumulates, which could titrate the cap-binding translation initiation factor,
eIF4E, away from translated mRNAs
•X29 (Nudt16) was identified in Xenopus as specific decapping enzyme for
U8 snoRNA. It is the main decapping activity in mammalian cells.
30
Eukaryotic decapping proteins
In yeast, major cytoplasmic mRNA decay mechanism is:
1) deadenylation
2) decappinng
3) 5’  3’ exo digestion
(can also detect minor 3’  5’ degradation pathway)
In mammals, major cytoplasmic mRNA decay mechanism is:
1) deadenylation
2) 3’  5’ digestion by exosome
3) decapping by scavenger decapping enzyme
(can also detect minor 5’  3’ degradation pathway)
31
Strategy to isolate mutations affecting
mRNA decay
*
probe
Northern
*pG cassette blocks exonucleases
32
mRNA decapping emzyme
yeast
human
Dcp1p
hDcp1a
hDcp1b
Dcp2p
Dcp2p
The holoenzyme responsible for decapping consists of two subunits in yeast and
mammals, Dcp1p and Dcp2p, with Dcp2p as the catalytic subunit
The role of Dcp1p seems to be to stimulate the activity of Dcp2p; Structural
data show that S. cerevisiae Dcp1p may stimulate Dcp2p activity, not through
mediation of RNA binding, but by changing Dcp2p conformation from an inactive
open state to an active closed one
Hydrolysis of the cap structure by Dcp1p–Dcp2p, or by Dcp2p alone, releases
m7GDP. This leaves an mRNA with a 5′ monophosphate, which is a preferred
substrate for the Xrn1p exonuclease.
Dcp1p–Dcp2p decapping enzyme recognizes the mRNA substrate by interactions
with both the cap and the RNA moiety and this is prevented by assembled
33
translation initiation complex
DCP2 is required for 5' to 3' mRNA decay
34
Dcp2p is required for 5' to 3' decay
following deadenylation
The stabilization of oligoadenylated, full-length mRNA species indicated
that 5' to 3' mRNA decay in the dcp2 mutant was blocked following
deadenylation
35
MFA2pG mRNA is stabilized in the dcp1-1
mutant
pGAL-MFA2pG
glu
glu
36
DCP2 overexpression specifically suppresses
the mRNA decay defect of the dcp1-2
mutation
GAL::MFA2pG
Glu
37
Regulators of yeast mRNA decapping
38
While both yeast and mammalian Dcp2 are active in
vitro on their own, in vivo they require additional
decapping activators, known as enhancers of
decapping (EDCs)
39
Regulators of yeast mRNA decapping
Proteins that associate with the Dcp1:Dcp2 complex can facilitate its
recruitment and stimulate mRNA decapping:
40
Decapping regulators bind to Dcp2p but differ in
mechanisms of decapping enhancement
Dhh1p and Pat1p repress translation directly, which enhances decapping because
translation and decapping are in competition.
Edc1p-3p and Pat1p are able to stimulate Dcp2p catalytic activity directly,
Pat1p serves as a scaffold for recruitment of other proteins, including Lsm1p-7p
heptamer. In agreement with its multiple roles in the regulation of decapping, Pat1p
deletion induces the most severe decapping defect among known yeast EDCs.
Lsm1p-7p complex forms a ring, binds to shortened poly(A) tail after
deadenylation and enhances interaction of Dcp2p with mRNA. Lsm1p-7p together
with Pat1p also bind to uridine stretches near the 3′ end of transcript, explaining
why Lsm1-7/Pat1 complex stimulates decapping following both mRNA deadenylation
and uridylation.
Xrn1 ribonuclease interacts with EDCs and this provides direct connection between
decapping and 5’ -3’ degradation.
Generally, Dcp1/Dcp2 are deposited on mRNAs as parts of a
ribonucleoprotein complex, and the composition of this complex varies in
different organisms.
41
decapping complexes recruit Dcp1–Dcp2p
to different mRNA substrates
42
There are at least two separate Lsm protein
complexes
Lsm1-7 complex
Degradation of NMD-targeted mRNAs
In mammals, rapid degradation of NMD targets can be achieved through at least
three different pathways
1.
SMG6
cleaves
mRNA
endonucleolytically in
the vicinity of PTCs and in human cells
the majority of NMD targets are
apparently
degraded
through
this
pathway. After SMG6-mediated cleavage,
for which SMG6 needs to interact with
UPF1, the 3’ RNA fragment is rapidly
degraded by XRN1 and the 5’ fragment
seems to be digested by the exosome.
2. The heterodimer SMG5–SMG7, which interacts with phosphorylated UPF1, Interacts
the deadenylase CNOT8 (POP2) interacts with this C-terminal of SMG7, indicating that
SMG5-SMG7 recruits the CCR4-NOT complex to NMD targets to induce their
deadenylation-dependent decapping and subsequent XRN1-mediated degradation.
Consistently, SMG7-mediated mRNA decay requires the presence of DCP2 and XRN1
but not of SMG6.
3. UPF1 also associates with decapping complex subunits DCP1A, DCP2, and
PNRC2, indicating that direct deadenylation-independent decapping might
constitute a third route to degrade aberrantly terminating mRNAs
Decapping complexes
45
The RPS28B mRNA contains a unusually
long 3′UTR
Rps28B
Rps28A
46
The RPS28B mRNA 3′UTR Contains a
cis Regulatory Element
Edc3-dependent increase of RPS28B stability depends on its 3’-UTR
mRNA levels
LacZ activity
pRPS28b
placZ+ 3UTR PRPS28b
47
A Model for the Edc3-Mediated Autoregulation of
the RPS28B mRNA
RPS28B mRNA is translated to produce Rps28b protein that are usually
assembled into ribosomal subunits. However, once the Rps28b protein
concentration reaches a certain threshold, the excess Rps28b molecules that
are produced bind to Edc3 within the Dcp1/Dcp2 complex. Rps28b binding to
Edc3 promotes Edc3 dimerization and leads to the formation of an active
decapping complex. This active decapping complex possesses unique substrate
specificity for RPS28B mRNA, binds to the RPS28B 3′ UTR decay-inducing
element, and catalyzes the removal of the cap structure from the RPS28B
mRNA.
48
mRNA Decapping Enzymes in Mammalian
Cells
49
Eukaryotic decapping proteins
(Nudt16)
NUDIX domains are essential
for enzymatic activity
50
Nudt16 Is a Cytoplasm mRNA Decapping
Enzyme
293T cell line transfected
with FLAG-Nutd16
51
Dcp2 and Nutd16 expression
Dcp2
Nudt16
52
Nudt16 Is Involved in the Decay of a
Subset of mRNAs
MEF cells were infected with lentivirus carrying shRNA against Nutd16 or
Xrn1
MEF
Dcp2
Transcription was arrested by the addition of Actinomycin D 48 hrs post
lentiviral infection and mRNA levels tested at the times indicated.
53
mRNA Decapping Enzymes in Mammalian
Cells
54
Uridylation by TUT4 and TUT7
Marks mRNA for Degradation
Over the past few years it has become evident that not only deadenylation, but
also extension of the 3’ -ends of protein-coding transcripts with stretches of
uridine residues, i.e. uridylation, may serve as an initial signal triggering mRNA
decay in the cytoplasm.
The human genome encodes 7 non-canonical RNA nucleotidyltransferases, with some
of them preferentially adding uridine instead of adenine, functioning more as
terminal uridyltransferases (TUTases) or poly(U) polymerases (PUPs)
Uridylating enzymes have been found in all eukaryotes, with the exception of S.
55
cerevisiae
Uridylation-dependent mRNA decapping
That uridylation may play
a prominent role in the
control of poly(A)+ mRNA
stability in the cytoplasm
was first demonstrated
from studies in S. pombe
with, Cid1 identified as
the first non-canonical
nucleotidyltransferase
Evolutionary conserved mechanism
mRNA stability increases substantially in
a cid1 deletion strain
oligouridylation occurs prior to decapping
57
Uridylation is largely Cid1 dependent and
precedes decapping
It was postulated that uridylation-dependent decay may be of particular
importance in S. pombe , since poly(A) tails present on its mRNAs are considerably
shorter than in other eukaryotes .
58
TUTase-4 (ZCCHC11) and TUTase-7 (ZCCHC6) proteins, non-canonical
nucleotidyltransferases homologous to fission yeast Cid1, were identified as
enzymes responsible for the uridylation of mRNA 3’ -ends in human cells. In
concordance with previous findings, siRNA-mediated
Gene-level analyses revealed that the majority of mRNA species (638 out
of 746 genes, 85.5%) are decreased in uridylation following TUT4/7
knockdown (p = 7.69 × 10−100, one-tailed Mann-Whitney U test) (Figure
1D; Table S1). This result strongly indicates that TUT4/7 uridylate most,
if not all, mRNAs
59
U-rich extensions were added more efficiently to shortened poly(A)
tails by TUTase-4/7 both in vivo and in vitro
in human cells, in contrast to S. pombe (possessing intrinsically
shorter poly(A) tails), mRNA deadenylation precedes uridylation
60
Dis3l2 is the central player of the novel,
exosome independent mRNA decay pathway in
the cytoplasm.
Dis3l2 is absent from S. cerevisiae , but was shown to participate in mRNA
degradation in S. pombe plants and human cells,,
Recent studies carried out in both S. pombe and mammalian cells revealed that
Dis3l2 nuclease, a paralogue of the exosome complex Dis3/Dis3l catalytic
subunits, works on its own preferentially degrading 3’ -uridylated mRNAs in the 3’
-5’ direction
In addition to activation of decapping and 5’ -3’ degradation, uridylation
apparently also stimulates mRNA decay in the other direction by enhancing
61
Dis3l2 exoribonuclease activity
62
TUTase-4 is the most likely enzyme involved in
histone mRNA decay
63
Processing (P) bodies
The exit of a mRNA from translation and
decapping seems to correlate with the entry
specific cytoplasmic foci, referred to as
mRNA decapping factors and the 5′→3′
concentrated in both yeast and mammals
its targeting for
of the mRNA into
P-bodies, where
exonuclease are
Decapping factors and Xrn1p localize to
discrete foci in the cell
Processing (P) Bodies
Alteration of mRNA decay affects the
size and the number of P bodies
GFP-Dhh1
the size and number of Pbodies varies in a manner that
correlates with the flux of
mRNA molecules through the
decapping step.
inhibiting mRNA decay at the
deadenylation step in a ccr4Δ strain
or inhibiting decapping by removing
the decapping activator Pat1p leads
to a reduction in P-body size and
number.
P bodies increase when the
decapping or 5’->3' exonucleolytic
digestion is inhibited
Dis3L2 co‐localizes with P‐bodies
68
P bodies components
1. Core Components:
mRNA +
P bodies components
2.Additional Components
miRNA repression factors
(metazoa)
yeast
yeast
RNA-Binding Proteins and
Translation Repressors
P bodies components
3. Proteins involved in nonsense-mediated decay (NMD)
yeast
human
yeast
yeast
only observed in P bodies
under
certain
mutant,
stress, or overexpression
conditions
human
human
These proteins may normally rapidly transit through P bodies
but under some conditions accumulate to detectable levels