B R I E F C O M M U N I C AT I O N S
Nonsense-mediated mRNA
decapping occurs on polyribosomes
in Saccharomyces cerevisiae
© 2010 Nature America, Inc. All rights reserved.
Wenqian Hu, Christine Petzold, Jeff Coller & Kristian E Baker
Nonsense-mediated decay (NMD) degrades mRNA containing
premature translation termination codons. In yeast, NMD
substrates are decapped and digested exonucleolytically
from the 5` end. Despite the requirement for translation in
recognition, degradation of nonsense-containing mRNA is
considered to occur in ribosome-free cytoplasmic P bodies.
We show decapped nonsense-containing mRNA associate with
polyribosomes, indicating that recognition and degradation
are tightly coupled and that polyribosomes are major sites for
degradation of aberrant mRNAs.
Cells use intricate mechanisms to ensure fidelity of gene expression
and to safeguard against the accumulation of aberrant mRNAs and
proteins. In eukaryotes, mRNAs harboring a premature translation
termination codon (PTC, or nonsense codon) within their proteincoding regions are efficiently detected and rapidly degraded1–4.
This surveillance pathway, termed nonsense-mediated mRNA decay
(NMD), prevents the accumulation of truncated polypeptides that
would potentially be toxic to the cell. NMD targets a heterogeneous
class of mRNAs, including those that have acquired a PTC as a consequence of gene mutation or errors during transcription. Moreover,
inaccurate or alternative pre-mRNA splicing generates a major subset
of PTC-containing mRNAs, and unproductively rearranged immunoglobulin and T-cell receptor genes represent an important physiological class of NMD substrates1. NMD also modulates the levels of
~1–10% of cellular mRNA lacking a PTC, thereby serving an additional role in regulating gene expression1,2.
Three phylogenetically conserved proteins, up-frameshift protein 1
(UPF1), UPF2 and UPF3, constitute the core of the NMD machinery and
are required for both the recognition of nonsense-containing mRNA and
their targeting for rapid degradation1,2. In the budding yeast Saccharomyces
cerevisiae, destruction of NMD substrates is initiated by removal of the
5` cap (decapping) followed by 5`l3` exonucleolytic digestion5. In mammals,
NMD triggers mRNA decapping and 5` digestion but also targets substrates
for simultaneous degradation at the 3` end catalyzed by the cytoplasmic
exosome6. Moreover, NMD targets in human cells7 can be cleaved endonucleolytically at a position proximal to the PTC, similar to the predominant pathway for degradation observed in Drosophila melanogaster cells1.
Recognition of premature termination by the NMD pathway requires
mRNA translation3. Consistent with this, both the NMD machinery and
its substrates are found associated with polyribosomes8,9. Degradation
of nonsense-containing mRNA, in contrast, is hypothesized to occur
after dissociation of the mRNA from ribosomes and in cytoplasmic foci
known as P bodies10,11. In support of this, UPF1 inhibits translation
of PTC-containing mRNA and also functions as a repressor of normal
mRNA translation10–12. Moreover, the decapping holoenzyme (DCP1
and DCP2), 5`l3` exoribonuclease (XRN1), UPF proteins and NMD
substrates localize to P bodies, proposed sites for mRNA storage and
decay10. Physical interactions observed between UPF1 and the decapping
enzyme (DCP2) are hypothesized to mediate targeting of NMD substrates to P bodies3,11. Based on these and additional observations, UPF1
is proposed to translationally repress and target the aberrant mRNA to P
bodies, where UPF2 and UPF3 activate decapping of the mRNA10.
We have recently shown that decapping and 5`l3` degradation of
normal mRNA in yeast occurs co-translationally and that dissociation
of mRNA from ribosomes is not a prerequisite for its destruction 13.
Based on these findings, we hypothesized that decapping of
nonsense-containing mRNA mediated by the NMD pathway also
occurs while the mRNA is associated with polyribosomes. Consistent
with a view that NMD substrates are degraded co-translationally,
NMD can be uncoupled from the visible aggregation of P bodies in
yeast, Drosophila and mammalian cells14–17.
A model for NMD featuring repression of mRNA translation would
predict that in cells lacking mRNA decapping activity, substrate mRNA
should be stabilized but should not be found bound to ribosomes. The
association of an efficient substrate for NMD in yeast, GFPPTC67 mRNA
(Supplementary Table 1, Supplementary Data and Supplementary Fig. 1),
with a translating messenger ribonucleoprotein (mRNP) was monitored
by sucrose density gradient sedimentation. Quantitative RT-PCR
(qRT-PCR) was used to assess mRNA abundance in gradient fractions
representing the nontranslating cellular RNA (RNP), monoribosomes
(80S) and polyribosomes (Fig. 1a,b). In wild-type (WT) cells, the vast
majority of GFPPTC67 mRNA was detected in light fractions (such as RNP),
with very little mRNA co-sedimenting with polyribosomes (Fig. 1c). For
cells lacking UPF1, the NMD factor implicated in translational repression10–12, increased levels of GFPPTC67 mRNA were detected and the
bulk of mRNA co-sedimented with polyribosomes, consistent with its
stabilization and continued translation in the absence of NMD. Similarly,
GFP mRNA lacking a PTC was stable and polysome associated (Fig. 1c).
Notably, GFPPTC67 mRNA observed in the absence of UPF1 in heavy gradient fractions represents bona fide polyribosomes and not an association
with some other dense particle (see below).
The diminished levels of GFPPTC67 mRNA levels associated with
polyribosomes for WT cells versus cells lacking UPF1 is not inconsistent
Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, Ohio, USA. Correspondence should be addressed to K.E.B. ([email protected]) or
J.C. ([email protected]).
Received 22 July 2009; accepted 13 November 2009; published online 31 January 2010; doi:10.1038/nsmb.1734
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a
RNP
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© 2010 Nature America, Inc. All rights reserved.
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Figure 1 NMD substrates are bound by polyribosomes when mRNA decapping is inhibited. (a) Polyribosome analysis of WT, upf1$ and dcp2$ cells.
RNPs, 80S ribosomes and polyribosomes are indicated. (b) RT-PCR analysis for GFPPTC67 mRNA is quantitative for the analysis shown in c.
(c) Distribution of GFP mRNA (lacking a premature termination codon, − PTC) and GFPPTC67 mRNA (harboring a premature termination codon, + PTC)
in sucrose-gradient fractions. Arbitrary levels of GFPPTC67 mRNA are compared for WT, upf1$ and dcp2$ cells. (d) Northern blot analysis to monitor
the sedimentation in sucrose gradients of CYH2 pre-mRNA and PGK1PTC225 reporter mRNA in WT, upf1$ and dcp2$ cells. The abundance of mRNA in
each fraction in terms of the percentage of the total is shown.
with inhibition of mRNA translation and degradation of the mRNA
once it is ribosome-free. However, the observation is also compatible
with destruction of the mRNA while associated with the translating
mRNP, where PTC recognition occurs. To discriminate between these
possibilities and determine whether GFPPTC67 mRNA is removed from
polyribosomes by the NMD machinery before its decay, we performed
polyribosome analysis on cells defective in mRNA decapping (dcp2$).
In these cells, NMD substrates are recognized by the NMD machinery
and are observed to be translationally inhibited but are not destabilized due to the absence of cellular decapping activity12. In dcp2$ cells,
GFPPTC67 mRNA levels were elevated, with a majority of the mRNA
co-sedimenting with polyribosomes (dcp2$; Fig. 1c). Unexpectedly,
the sedimentation pattern of GFPPTC67 mRNA was indistinguishable
between decapping-defective cells (dcp2$) and those inhibited for
NMD (upf1$), and, notably, was not enhanced in the RNP in dcp2$
cells (Fig. 1c). These data suggest that in the absence of decapping,
NMD substrates are not dissociated from polyribosomes by UPF1
but rather remain bound by ribosomes. Consistent with qRT-PCR
analysis of GFPPTC67 mRNA, additional NMD substrates (such as
PGK1PTC225 reporter mRNA and the endogenous CYH2 pre-mRNA)
were found by northern blotting to be associated with polyribosomes
in dcp2$ cells, and their sedimentation patterns corresponded to the
length of the coding region (Fig. 1d). Collectively, these data indicate
that UPF1 does not promote a ribosome-free mRNP and support a
view that NMD substrates are degraded while still associated with
polyribosomes.
To evaluate whether bulk levels of decapped nonsense-containing
mRNA can be found associated with polyribosomes, we performed
quantitative primer extension analysis on sucrose-gradient fractions
from xrn1$ cells, where the products of decapping are enriched 13.
We detected the majority (~80%) of decapped PGK1PTC225 reporter
mRNA on polyribosomes (Supplementary Fig. 2). To examine
2
whether the decapping of NMD substrates occurs coincidentally with
the mRNA’s association with ribosomes, we used splinted ligation followed by RT-PCR to detect products of the decapping reaction that
are short-lived in WT cells13. In this procedure, an RNA adaptor is
ligated to the 5` end of a decapped mRNA (assisted by a DNA splint)
and the intermediate used as a template for cDNA synthesis and
amplification of a DNA product by PCR (Fig. 2a). Decapped products
of ‘normal’ (for example, RPL41a mRNA) and nonsense-containing
mRNA (endogenous CYH2 pre-mRNA and PGK1PTC225 mRNA) were
detected in WT cells but not in dcp2$ cells, where mRNA decapping is
inhibited, showing that product formation is dependent on decapping
in vivo (Fig. 2b, lane 1 versus lane 3). Consistent with this, removal of
the mRNA 5` cap in vitro by tobacco acid pyrophosphatase resulted
in detection of decapped mRNA from dcp2$ cells (Fig. 2b, lane 4).
Notably, decapping of PGK1PTC225 mRNA required NMD, as products
were greatly reduced in cells lacking UPF1 (Fig. 2c). Splinted ligation
analysis of RNA recovered from sucrose-gradient fractionation of
WT cells established that the bulk of decapped CYH2 pre-mRNA
and PGK1PTC225 mRNA co-sedimented with polyribosomes (Fig. 2d).
Critically, the sedimentation pattern of decapped mRNA correlated
with the open reading frame length of these two mRNAs, illustrating that sedimentation was a result of polyribosome association.
Specifically, the product of unspliced CYH2 pre-mRNA (encoding
an 18-residue polypeptide) associated predominantly with 80S and
light polyribosome regions, whereas PGK1PTC225 mRNA (encoding a
224-residue polypeptide) was detected deep in the gradient and with
high-density polyribosomes (Fig. 2d). Given the transient nature of
uncapped mRNA in WT cells, we interpret the association of these
decay intermediates with polyribosomes as a consequence of their
co-translational generation.
The decapping of mRNA represents the critical, rate-determining
step in the degradation of both normal and NMD substrates, and we
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B R I E F C O M M U N I C AT I O N S
a
7mGppp
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© 2010 Nature America, Inc. All rights reserved.
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15
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Figure 2 The association of decapped NMD substrates with polyribosomes. (a) The splinted ligation RT-PCR assay for detecting decapped mRNA.
(b) Detection of decapped CYH2 pre-mRNA, PGK1PTC225 mRNA and RPL41A mRNA in WT, dhh1$/pat1$ and dcp2$ cells. Addition of tobacco alkaline
phosphatase before splinted ligation RT-PCR (dcp2$ + TAP), without splinted ligation step (− Ligation), without reverse transcription step (− RT) and
without cDNA template added to the PCR reaction (− cDNA). (c) Detection of decapped PGK1PTC225 mRNA in WT and upf1$ cells. (d,e) Splinted
ligation RT-PCR analysis on RNA recovered from sucrose-gradient fractionation of wild-type (d) or dhh1$/pat1$ (e) cells. RNPs, 40S and 60S ribosomal
subunits, 80S ribosomes and polyribosomes are indicated. The abundance of mRNA in each fraction in terms of the percentage of the total is shown.
have shown that normal mRNA decapping occurs co-translationally13.
To ensure that the accumulation of decapped PGK1PTC225 mRNA on
polyribosomes was due to recognition and targeting by the NMD
pathway and was not a consequence of shunting of the mRNA into
the deadenylation-dependent pathway of decay used for the metabolism of normal mRNA, we performed splinted ligation analysis
on cells in which decapping of normal but not PTC-containing
mRNA is defective. Deletion of the activators of mRNA decapping,
DHH1 and PAT1, leads to marked mRNA stabilization and
accumulation of deadenylated, capped intermediates for normal but
not nonsense-containing mRNA18 (Supplementary Figs. 3 and 4).
Consistent with this, analysis of mRNA decapping by splinted
ligation RT-PCR failed to detect decapped products for RPL41a
mRNA in dhh1$/pat1$ cells as compared to WT cells (Fig. 2b).
In contrast, the products of decapping were readily detected for
two nonsense-containing mRNAs in dhh1$/pat1$ cells (CYH2
pre-RNA and PGK1PTC225; Fig. 2b). Moreover, destabilization of PTCcontaining mRNA in dhh1$/pat1$ cells required NMD as demonstrated by its dependence on UPF1 (Supplementary Fig. 4d).
These data establish that decapping of nonsense-containing
mRNA in dhh1$/pat1$ cells occurs as a consequence of targeting by NMD. We therefore evaluated the sedimentation pattern of
decapped CYH2 pre-mRNA and PGK1PTC225 mRNA by sucrosegradient analysis using splinted ligation RT-PCR in dhh1$/pat1$
cells. Notably, decapped products of nonsense-containing mRNA
from these cells sedimented in a similar manner to that observed
for WT (Fig. 2d versus e). We conclude that polyribosome association of the decapped products of NMD substrates observed in both
WT and dhh1$/pat1$ mutant cells are the result of decapping triggered by NMD.
To further demonstrate the association of decapped intermediates
of nonsense-containing mRNA with ribosomes, we purified ribosomes from cell extracts before assaying for decapped RNA products19.
Immunoprecipitation of affinity-tagged ribosomal protein RPL25
co-purified 25S and 18S ribosomal RNA and ribosome-bound
mRNA (such as CYH2 mRNA) but not nontranslated RNA (such
as U1 snRNA) as detected by northern blotting (Fig. 3). Critically,
splinted ligation RT-PCR analysis showed that decapped products
of CYH2 pre-mRNA, an endogenous NMD substrate, also purified
with ribosomes (Fig. 3). Notably, a similar product was not amplified
from cells lacking epitope-tagged RPL25 protein. The co-purification
of decapped nonsense-containing mRNA with ribosomes supports a
mechanism in which the first step in the degradation of NMD substrates occurs co-translationally.
In summary, we have presented evidence that decapping of nonsense-containing mRNA by NMD occurs while the substrate is bound
by ribosomes. Our findings are consistent with the association of the
NMD factors UPF1, UPF2 and UPF3 with polyribosomes8,9, as well
as DCP2 and XRN1, the catalytic activities for decapping and 5`l 3`
exonucleolytic RNA digestion9,13,20. Moreover, the rapid degradation of
NMD substrates upon removal of cycloheximide, an inhibitor of translation elongation, also supports a co-translational decay mechanism4.
Our data indicate that dissociation of nonsense-containing mRNA
from ribosomes does not occur before their decay, which is inconsistent
with their sequestration away from polyribosomes followed by targeting and destruction in P bodies. In agreement with this, the rapid decay
of NMD substrates can be uncoupled from detectable P-body aggregation in several organisms, including yeast14–17. Notably, endonucleolytic cleavage of NMD substrates proximal to the PTC observed in
Drosophila and human cells suggests that the prematurely terminating
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B R I E F C O M M U N I C AT I O N S
UPF3) or whether the kinetics of 5`-end remodeling and decapping
are much faster than the rate of ribosomal run-off once translational
repression ensues.
Flag-RPL25
IP: antiFlag
RPL25
Flag-RPL25
RPL25
Input
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
Decapped
CYH2 pre-mRNA
ACKNOWLEDGMENTS
The authors thank members of the Baker and Coller laboratories for critical
evaluation of the manuscript. W.H. is supported by the American Heart
Association. C.P. is supported by an undergraduate stipend provided by the US
National Science Foundation. Funding was provided by the US National Institutes
of Health (GM080465).
CYH2 mRNA
U1 snRNA
25S rRNA
© 2010 Nature America, Inc. All rights reserved.
18S rRNA
Figure 3 Decapped products of nonsense-containing mRNA co-purify with
ribosomes. Immunoprecipitation of ribosomes (mediated by affinity-tagged
large ribosomal protein, RPL25) co-purifies decapped CYH2 pre-mRNA
as detected by splinted ligation and RT-PCR analysis. Co-purification of
CYH2 mRNA and ribosomal RNA (rRNA) were detected by 1.4% agarose
gel followed by northern blot and ethidium bromide staining, respectively.
U1 snRNA did not co-purify with ribosomes as monitored by northern blot.
ribosome helps in delineating the site of the nonsense codon and that
these substrates are also degraded while ribosome associated.
Although our observations indicate that recognition and decay of
nonsense-containing mRNA by NMD are tightly coupled, they raise
several mechanistic questions. Of particular interest are recruitment
of the decapping complex and how the mRNA is rapidly decapped,
particularly in light of its association with the translation machinery.
It has been previously observed that translation of PTC-containing
mRNAs is inhibited and that UPF1 functions as a general translational
repressor10–12. In the context of co-translational decapping, it will
be important to determine whether UPF1 acts to inhibit translation
elongation or to facilitate rapid rearrangement of translational initiation factors at the mRNA 5` end, thereby allowing the decapping
enzyme accessibility to the cap. Our finding that NMD substrates
associate with polyribosomes in dcp2$ cells supports a mechanism
of translation inhibition downstream of initiation; however, UPF1
interacts with eIF3, a critical factor in 80S ribosomal-subunit joining11. To reconcile these observations, it will be necessary to determine
whether ribosomes associated with NMD substrates are impaired
in elongation in response to the action of UPF1 (and/or UPF2 and
4
AUTHOR CONTRIBUTIONS
W.H., J.C. and K.E.B. designed experimental strategies; W.H. and C.P. performed
experimental analysis and interpreted data; J.C. and K.E.B. interpreted data and
wrote the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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Supplementary information for:
Nonsense-mediated mRNA decapping occurs on polyribosomes in Saccharomyces cerevisiae
Wenqian Hu, Christine Petzold, Jeff Coller*, and Kristian E. Baker*
Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106
*To whom correspondence should be addressed: K. Baker ([email protected]) or
J. Coller ([email protected])
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
a
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Supplementary Figure 1 GFP PTC67 mRNA is a substrate for nonsense-mediated mRNA decay.
(a) Transcription shut-off analysis of GFP PTC67 reporter mRNA in wild-type and upf1! cells.
RNA (30 !g) isolated from cell aliquots removed at various time points after inhibition of
transcription were subject to Northern blot analysis. GFP PTC67 mRNA and a loading control, scR1
RNA, were detected using radiolabeled complementary oligonucleotides. (b) Quantification of mRNA
levels, after normalization, measured in (a).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
a
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Supplementary Figure 2 Decapped, nonsense-containing mRNA is associated with polyribosomes in
xrn1! cells. (a) Primer extension analysis for PGK1 PTC225 mRNA was performed on RNA recovered
from sucrose gradient fractions after centrifugation of lysates from xrn1! cells. RNP, 80S, and
polyribosome sedimentation profiles are indicated. FL: full length mRNA; – cap: decapped mRNA.
(b) Quantification of decapped PGK1 PTC225 mRNA in RNP versus polyribosome fractions.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
WT
(min)
A
m7Gppp AUG
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(55-75)
An
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A
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Supplementary Figure 3 dhh1!/pat1! cells exhibit a strong defect in decapping of normal mRNA.
Transcription pulse-chase analysis for MFA2pG reporter mRNA in wild-type and dhh1!/pat1! cells.
Reporter gene expression was induced for 7 minutes and followed by inhibition of transcription.
RNA, isolated from time points after inhibition of transcription, was analyzed by high resolution
polyacrylamide gel electrophoresis and Northern blotting. MFA2pG reporter mRNA was detected
using an oligonucleotide complementary to the poly(G) tract in the 3’ UTR. scR1 RNA served as a
control for RNA loading. PI: pre-induction; dT: RNA treated with oligo d(T)/RNase H to delineate the
migration pattern of deadenylated mRNA.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
"
p2
dc
dh
up
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"
h1
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"
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pre-mRNA
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Time (min):
0
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25
d
G hh1
AL "
1: /pa
:U t1
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CYH2 pre-mRNA
20
CYH2 mRNA
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p
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at
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Supplementary Figure 4 Degradation of nonsense-containing mRNA is unaffected in dhh1!/pat1! cells
and dependent upon NMD. (a) Steady-state CYH2 pre-mRNA levels in wild-type, upf1!, dhh1!/pat1!,
and dcp2! cells were detected by Northern blot. scR1 RNA levels serve as a control for loading.
(b) Transcription shut-off analysis of PGK1 PTC225 mRNA in wild-type, upf1!, dhh1!/pat1!, and dcp2!
cells. PGK1 PTC225 mRNA half-lives after normalization to scR1 RNA (c). (d) Steady-state CYH2 mRNA
and pre-mRNA levels in wild-type, upf1!, and dhh1!/pat1!/GAL1::UPF1 cells (UPF1 gene under control
of the galactose-inducible, GAL1 promoter). Gal: cell grown in galactose media; Glu: cells grown in
glucose media.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
Table 1. Yeast Strains, Plasmids and Oligonucleotides
Name
Description
yJC151
MATa, ura3!, leu2!, his3!, met15!
EUROSCARF
Reference
yJC182
MATa, ura3!, leu2!, his3!, met15!, xrn1::KanMX6
EUROSCARF
yJC287
CB012: MATa, ade2-1, his3, leu2, trp1, ura3, pep4::HIS3, prb::HIS3, pre1::HIS3
yJC288
YIT613: MATa, ade2-1, his3, leu2, trp1, ura3, pep4::HIS3, prb::HIS3, pre1::HIS3, rpl25::LEU2 [pRPL25-Flag-URA3-CEN]
yJC324
MATa, ura3!, leu2!, his3!, met15!, dhh1::KanMX6, pat1::HIS3
yJC327
MATa, ura3!, leu2!, his3!, met15!, dcp2::KanMX6
2
yJC443
MATa, ura3!, leu2!, his3!, met15!, upf1::KanMX6
EUROSCARF
yJC751
MATa, ura3!, leu2!, his3!, met15!, dhh1::KanMX6, pat1::HIS3, URA3::GAL1-HA-UPF1
1
1
This study
This study
pKB290 GFP WT
3
pKB303 GFP-PTC at codon 67
3
pJC331
PGK1pG
This study
pJC364
PGK1-PTC225
This study
pRP469
MFA2pG
oJC591
5'-CGGATAAGAAAGCAACACCTGGC-3'
This study
oJC620
5'-GATCAATTCGTCGTCGTCGAATAAAGAAGACAA-3'
This study
oJC652
5'-ACCAAGGAGTTTGCATCAATGAC-3'
This study
oJC706
5'-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3'
This study
oJC707
5'GCTGATGGCGATGAATGAACACTG-3'
This study
oJC809
5'-ACAAGATTCAATTGATTGACAACTAGTTGGACAAGGTCGACTCTATCATCA-3'
This study
oJC810
5'-TGATGATAGAGTCGACCTTGTCCAACTAGTTGTCAATCAATTGAATCTTGT-3'
This study
oJC826
5'-ATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTT TTTCATCAAAGCCAGCAAACGCAGTGTTCATTCATCGCCATCAGC-3'
This study
oJC834
5'-AAAGTCACCGTCTTGGTTCTTTCATTCCCT-3'
This study
oJC836
5'-TGGAAGGCATTCTTGATTAGTTGGATGA TTTCATCAAAGCCAGCAAACGCAGTGTTCATTCATCGCCATCAGC-3'
This study
oJC838
5'-GCTCTCATTTCGATTGAATCGATGTGGTCT TTTCATCAAAGCCAGCAAACGCAGTGTTCATTCATCGCCATCAGC-3'
2
oJC839
5'-CTGGCTCTCACCTTCCGTCTCTTTCTCTTA-3'
2
oKB118
5’-GGAGAAGAACTCTTCACTGGAGTTGTCCC-3’
This study
oKB132
5’-GGGCAGATTGTGTGGACAGGTAATGGTTGTCTG-3’
This study
oKB347
5’-CATAACCTTCGGGCATGGCACTCTTG-3’
This study
oRP100
5'-GTCTAGCCGCGAGGAAGG-3'
4
oRP121
5'-AATTCCCCCCCCCCCCCCCCCCA-3'
4
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
4
SUPPLEMENTARY DATA
GFPPTC67 reporter mRNA is degraded by NMD
To demonstrate GFP reporter mRNA containing a PTC at codon 67 (GFPPTC67) is targeted to
degradation by NMD, a transcription shut-off analysis was performed on wild-type and upf1!
cells (Supplementary Fig. 1). GFPPTC67 reporter mRNA was stabilized ~4-fold in upf1! cells (cf.
4 min versus 15 min in wild-type versus upf1! cells).
Decapping of normal mRNA is strongly inhibited in dhh1!/pat1! cells
Deletion of the decapping activators DHH1 and PAT1 leads to a defect in mRNA decapping and
a dramatic stabilization of normal mRNA18. To confirm that decapping of mRNA is inhibited in
dhh1!/pat1! cells, transcription pulse-chase analysis was performed for MFA2pG reporter
mRNA in wild-type and dhh1!/pat1! cells (Supplementary Fig. 2). Specifically, a brief
induction of reporter mRNA transcription followed by inhibition of transcription produces a
homogenous population of MFA2pG mRNA and decay of the mRNA is monitored over time. In
wild-type cells, MFA2pG mRNA is deadenylated prior to the disappearance of the full-length
mRNA and appearance of a decay fragment (generated by the block of 5’"3’ exonucleolytic
RNA digestion by the 18 nucleotide G track in the mRNA 3’ UTR)18. In dhh1!/pat1! cells,
reporter mRNA is deadenylated, however, the deadenylated mRNA is stable and accumulates
over the time course and no decay fragment is detected. The stabilization of deadenylated
mRNA is indicative of a defect in mRNA decapping.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734
Degradation of NMD substrates is unaffected in dhh1!/pat1! cells
Three lines of evidence were used to demonstrate that recognition and degradation of NMD
substrates are not affected in dhh1!/pat1! cells. First, the steady-state level of the endogenous
NMD substrate, CYH2 pre-mRNA, was unchanged in dhh1!/pat1! cells (as compared to wildtype cells; Supplementary Fig. 3a). In contrast, CYH2 pre-mRNA levels increased in both NMD
mutants (upf1!) and decapping defective cells (dcp2!). Second, transcription shut-off analysis
for a PTC-containing reporter (PGK1PTC225 mRNA) revealed that the mRNA half-life was
identical in wild-type versus dhh1!/pat1! cells (Supplementary Fig. 3b & c). Inhibition of NMD
(upf1!) or mRNA decapping (dcp2!) lead to dramatic stabilization of PGK1PTC225 mRNA,
demonstrating that the reporter is indeed sensitive to NMD and degraded by a decappingdependent mechanism. Third, when UPF1 was depleted in dhh1!/pat1!/UPF1::GAL1 cells, the
level of CYH2 pre-mRNA increased dramatically (Supplementary Fig. 3d), indicating that
degradation of nonsense-containing mRNA is dependent upon UPF1 and decayed predominantly
by NMD in dhh1!/pat1! cells. Together, these results demonstrate that the degradation of
nonsense-containing mRNA in dhh1!/pat1! cells is unaffected and dependent upon NMD.
SUPPLMENTARY LITERATURE CITED
1!
Inada, T. et al. RNA 8, 948-958 (2002).
2!
Hu, W., Sweet, T.J., Chamnongpol, S., Baker, K.E., & Coller, J. Nature 461, 225-229
(2009).
3
4!
!
Baker, K.E. & Parker, R. RNA 12, 1441-1445 (2006).
Muhlrad, D., Decker, C.J., & Parker, R. Mol Cell Biol 15, 2145-2156 (1995).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1734