The RNA helicase Dhh1p cooperates with Rbp1p to promote porin mRNA decay via its non-conserved C-terminal domain

Article (PDF Available)inNucleic Acids Research 40(3):1331-44 · February 2012with23 Reads
DOI: 10.1093/nar/gkr803 · Source: PubMed
Abstract
The yeast RNA helicase Dhh1p has been shown to associate with components of mRNA decay and is involved in mRNA decapping and degradation. An RNA-binding protein, Rbp1p, is known to bind to the 3′-UTR of porin (POR1) mRNA, and induces mRNA decay by an uncharacterized mechanism. Here, we show that Dhh1p can associate with POR1 mRNA and specifically promote POR1 mRNA decay via its interaction with Rbp1p. As compared to its mammalian homolog RCK/p54/DDX6, Dhh1p has a unique and long extension at its C-terminus. Interestingly, this non-conserved C-terminal region of Dhh1p is required for interaction with Rbp1p and modulating Rbp1p-mediated POR1 mRNA decay. Notably, expression of a C-terminal 81-residue deleted Dhh1p can fully complement the growth defect of a dhh1Δ strain and retains its function in regulating the mRNA level of an RNA-binding protein Edc1p. Moreover, mammalian DDX6 became capable of interacting with Rbp1p and could confer Rbp1p-mediated POR1 mRNA decay in the dhh1Δ strain upon fusion to the C-terminal unique region of Dhh1p. Thus, we propose that the non-conserved C-terminus of Dhh1p plays a role in defining specific interactions with mRNA regulatory factors that promote distinct mRNA decay.
6 Figures

Full-text (PDF)

Available from: Fang-Jen Lee
Other full-text sources
The RNA helicase Dhh1p cooperates with
Rbp1p to promote porin mRNA decay via
its non-conserved C-terminal domain
Lin-Chun Chang
1,2
and Fang-Jen S. Lee
1,2,
*
1
Institute of Molecular Medicine, College of Medicine, National Taiwan University and
2
Department of Medical
Research, National Taiwan University Hospital, Taipei 100, Taiwan
Received July 4, 2011; Revised August 30, 2011; Accepted September 12, 2011
ABSTRACT
The yeast RNA helicase Dhh1p has been shown to
associate with components of mRNA decay and is
involved in mRNA decapping and degradation. An
RNA-binding protein, Rbp1p, is known to bind to
the 30-UTR of porin (POR1) mRNA, and induces
mRNA decay by an uncharacterized mechanism.
Here, we show that Dhh1p can associate with
POR1 mRNA and specifically promote POR1 mRNA
decay via its interaction with Rbp1p. As compared
to its mammalian homolog RCK/p54/DDX6, Dhh1p
has a unique and long extension at its C-terminus.
Interestingly, this non-conserved C-terminal region
of Dhh1p is required for interaction with Rbp1p and
modulating Rbp1p-mediated POR1 mRNA decay.
Notably, expression of a C-terminal 81-residue
deleted Dhh1p can fully complement the growth
defect of a dhh1"strain and retains its function in
regulating the mRNA level of an RNA-binding
protein Edc1p. Moreover, mammalian DDX6
became capable of interacting with Rbp1p and
could confer Rbp1p-mediated POR1 mRNA decay
in the dhh1"strain upon fusion to the C-terminal
unique region of Dhh1p. Thus, we propose that the
non-conserved C-terminus of Dhh1p plays a role in
defining specific interactions with mRNA regulatory
factors that promote distinct mRNA decay.
INTRODUCTION
Regulation of mRNA stability is an important step that
modulates the cellular abundance of a transcript (1). In
the yeast Saccharomyces cerevisiae, degradation of mRNA
occurs through two general pathways, both of which are
initiated by shortening of the poly(A) tail by the major
deadenylase complex. Following deadenylation, mRNA
can undergo 30–50exonucleolytic decay, which is catalyzed
by the exosome and the SKI complex. Alternatively, the
50-cap structure of an mRNA can be removed by the
decapping enzyme Dcp1p/Dcp2p, followed by 50–30cyto-
plasmic exonuclease Xrn1p digestion of the remainder of
the mRNA (2,3). Decapping is thought to be an irrevers-
ible step and a site of regulatory signal inputs (4). Several
proteins function in modulating decapping during mRNA
decay, including Dhh1p, Pat1p, Lsm1-7p complex and
Edc1/2p. These proteins are evolutionarily conserved
and each plays a distinct role in modulating the decapping
process (5–9).
One of these modulators of decapping, Dhh1p is a
member of the DEAD-box protein family of RNA
helicases. RNA helicases are ubiquitous, highly conserved
enzymes that participate in multiple aspects of RNA me-
tabolism, from transcription to degradation (10–13).
These proteins bind to and remodel RNA or RNA–
protein complexes in an ATP-dependent fashion (14).
Many RNA helicases are required for specific post-
transcriptional processes (11,13). However, in vitro assays
for RNA helicase activities performed with purified RNA
helicase proteins typically show little or no RNA sequence
specificity. Dhh1p contains nine conserved motifs charac-
teristic of DEAD-box proteins, which fold into two
RecA-like catalytic domains (11,15). Within the Dhh1p
helicase subfamily, there are significant differences related
to the extensions of the N- and C-termini of amino acid
sequences (16). For example, Dhh1p has a unique and
long extension at its C-terminus and a short extension at
its N-terminus. In contrast, the mammalian homolog
RCK/p54/DDX6 has a long N-terminal extension and
only a short C-terminal region. Therefore, it is thought
that these diverse regions could interact with specific pro-
tein complexes to provide target specificity of RNA
helicases in vivo (16,17). Dhh1p interacts with the decapp-
ing and deadenylase complexes (7) and along with other
proteins involved in decapping and mRNA degradation
localizes to discrete cytoplasmic foci known as processing
*To whom correspondence should be addressed. Tel: +8862 2312 3456 (Ext. 65730); Fax: +8862 2395 7801; Email: fangjen@ntu.edu.tw
Published online 13 October 2011 Nucleic Acids Research, 2012, Vol. 40, No. 3 1331–1344
doi:10.1093/nar/gkr803
ßThe Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
bodies (P-bodies) (18). P-bodies are speculated to be cyto-
plasmic locations of mRNA repression and decay. Reijns
et al. (19) have suggested that Dhh1p contains a
glutamine–proline-rich C-terminus and this region con-
tributes to efficient accumulation of Dhh1p in P-bodies
under stress conditions. Interestingly, in addition to the
implication of its function in mRNA decapping and
decay, Dhh1p homologs in Drosophila (Me31B),
Caenorhabditis elegans (cgh-1) and Xenopus (Xp54) have
been implicated in translational repression and storage of
maternal mRNA (20–23).
We have previously identified an RNA-binding protein
encoded by RBP1 (24) that can bind to and regulate the
stability of POR1 mRNA in S. cerevisiae (25). Rbp1p
contains three RNA recognition motifs (RRMs), two
glutamine-rich stretches and a C-terminal asparagine–
methionine–proline-rich (NMP) region. It binds to the
30-untranslated region (UTR) of POR1 mRNA through
its RNA recognition motifs and accelerates porin mRNA
turnover. Rbp1p-mediated POR1 mRNA decay is dis-
rupted in an xrn1Dstrain, indicating that POR1 mRNA
undergoes degradation through an Xrn1p-dependent
pathway following decapping (26). It is unclear whether
Rbp1p can recruit the general 50–30decay machinery to
promote POR1 mRNA decay. Using a yeast two-hybrid
assay, we have found that Dhh1p interacts with Rbp1p,
but the biological significance of such an interaction has
not characterized.
In this article, we provide evidence that Dhh1p cooper-
ates with Rbp1p to regulate a distinct mRNA decay. We
show that the non-conserved C-terminal region of Dhh1p
is required for interaction with Rbp1p and this interaction
is required for Rbp1p-mediated POR1 mRNA decay.
Moreover, the mammalian Dhh1p ortholog, DDX6,
which could neither interact with Rbp1p nor promote
POR1 mRNA decay, gains the ability to interact with
Rbp1p and elicit Rbp1p-mediated POR1 mRNA decay
in a dhh1strain when fused with the C-terminal
unique region of Dhh1p. Through our study, we propose
that Dhh1p is recruited to specific mRNAs and promotes
distinct mRNA decay by interacting with RNA-binding
protein complexes through its non-conserved C-terminal
region.
MATERIALS AND METHODS
Growth medium and yeast strains
Genotypes for the yeast strains used in this study are listed
in Supplementary Table SI. Yeast cells were grown either
in rich medium containing 1% yeast extract, 2% peptone
and 2% glucose or in synthetic media containing 0.67%
yeast nitrogen base (without amino acids) and 2% glucose
supplemented with the appropriate nutrients. Yeasts were
transformed by the lithium acetate method. The DHH1
gene was disrupted in YTC345 using a Kan disruption
cassette amplified by PCR from pFA6-kanMX6 (27).
Strains expressing Dcp2p-mCh, Dhh1p-GFP, Dhh1p-
dC81-GFP, Dhh1p-dC106-GFP, Dhh1p–3HA, Dhh1p-
dC81-3HA, or Rbp1p–3HA were obtained through
insertion of an mCherry-kan cassette amplified from
pBS34 (Roger Tsien lab), a GFP-HIS cassette amplified
from pFA6a-GFP(S65T)-His3MX6, or a 3HA-HIS
cassette amplified from pFA6a-3HA-His3MX6 (27).
Disruption or insertion of each cassette was verified by
western blotting.
Plasmid construction
Plasmids used in this study are listed in Supplementary
Table SII. Escherichia coli DH5awas used for DNA
manipulations. Plasmids were constructed by standard
protocols. For expression in yeast, the plasmid
pVT101U-HA-RBP1 was used, as previously described
(25), and HA-RBP1 containing the ADH1 promoter was
also cloned into YEplac181. The full-length DHH1,
DHH1-dC81 and DHH1-dC106 were amplified by PCR
from yeast genomic DNA using specific primers and
cloned into the pVT101U vector. These plasmids were
digested with restriction enzymes to isolate the DNA
fragment containing the ADH1 promoter and the open
reading frame. These fragments were subcloned into cor-
responding sites in YCplac111 to generate a series of
Dhh1p expression plasmids. The C85 PCR fragment from
the DHH1 gene, encoding the C-terminal 85 amino acids
of Dhh1p, was amplified using specific chimera primers;
with the 50-end aligned with DDX6 and the 30-end aligned
with DHH1. Full-length DDX6 and DDX6-C85 were
amplified by PCR from HeLa cDNA using specific
primers and C85 PCR fragments, and then cloned into
the expression vectors. For yeast two-hybrid assay,
full-length RBP1 and DHH1 were separately amplified
by PCR from yeast genomic DNA using specific primers
and cloned into the pJG4-5 vector in-frame with the
Gal4AD domain. Analogously, full-length and mutant
RBP1 or DHH1 were generated and cloned into
pEG202 in frame with the LexA DNA-binding domain.
For in vitro GST pull-down assays, the coding sequence
for full length and amino acid 1–425 of Dhh1p and for
C-terminal fragment (CF) of Rbp1p were amplified by
PCR from yeast genomic DNA with specific primers and
subcloned into the pET15b or pGEX4T-1 expression
vector, respectively.
Yeast two-hybrid assay
The yeast strain YEM1awas co-transformed with differ-
ent combinations of bait (pEG202) and prey (pJG4-5)
plasmids and b-galactosidase plate assays were performed
by streaking transformants onto SC-Trp-His plates con-
taining 2% galactose and 80 mg/ml X-Gal (5-bromo-
4chloro-3-indolyl-b-D-galactoside). The plates were then
incubated at 30C for 2–3 days.
Yeast cell extracts preparation and western blotting
Extracts were obtained from 2OD
600
of yeast cells, sus-
pended in 5% TCA and processed by vigorous vortexing
with glass beads. Cell debris was collected by centrifuga-
tion at 13 000 rpm for 10 min, washed with water
to remove residual TCA, followed by centrifugation at
13 000 rpm for 10 min, suspended in SDS-loading buffer
and then heated at 95C for 5–10 min. For western blott-
ing, all cell extracts were run on 9% SDS–polyacrylamide
1332 Nucleic Acids Research, 2012, Vol. 40, No. 3
gels. Proteins were then transferred to nitrocellulose
membrane and probed with indicated antibodies. Act1p
was used as a loading control.
In vitro GST pull-down experiments
Escherichia coli strain BL21 (DE3) (Novagen) was trans-
formed with plasmids pET15b-Dhh1p, pET15b-Dhh1p-
dC81, pGEX6T-1 or pGEX6T-1-Rbp1p-CF. Recombinant
GST, GST-Rbp1p-CF, His-Dhh1p and His-Dhh1p-dC81
were expressed in BL21 (DE3) by induction with 0.4 mM
isopropyl-1-thio-b-D-galactopyranoside (IPTG) at 25C
for 4 h. GST-fusion proteins or His-tagged proteins were
purified from E. coli lysates using glutathione–Sepharose
4B (GE Healthcare) or nickel-affinity resins (Qiagen), re-
spectively, according to the manufacturers’ instructions.
For the pull-down assays, GST or GST-Rbp1p proteins
were immobilized on glutathione agarose beads and
incubated with His-Dhh1p or His-Dhh1p-dC81 in
binding buffer (PBS, pH 7.4, 0.01% Triton X-100) for
2 h at 4C. The beads were collected and washed four
times with 1 ml of binding buffer. Bound protein was
then analyzed by 9% SDS–PAGE and western blotting
using anti-His monoclonal (BD Biosciences) and
anti-GST polyclonal antibodies.
Fluorescence microscopy
Cells were grown in YP-rich medium containing 2%
glucose to log phase. For glucose starvation, cells were
centrifuged and quickly washed with YP medium lacking
glucose and incubated for 20 min in a shaking incubator at
30C. Images were acquired with a Zeiss Axioskop micro-
scope (Germany) using a Plan-NeoFluar 100/1.32 ob-
jective and Cool snap fx CCD camera (Photometrics)
driven by Image-Pro Plus software. For quantification of
co-localization of Dhh1p-GFP or its mutants to P-bodies,
we randomly selected images, which contained at least 50
cells with Dcp2p-mCh foci in three independent experi-
ments. P-bodies were identified by the presence of
Dcp2p-mCh foci. For each GFP-fusion protein, 50 cells
with Dcp2p-mCh foci were counted the number of cells
with co-localization foci. The percentage of Dcp2p-mCh
foci containing cells showing GFP-fusion proteins in foci
after glucose starvation was determined. Data are repre-
sented as mean of three experiments SD.
Northern blotting and mRNA decay assay
For steady-state mRNA analysis, cells were grown in syn-
thetic medium lacking the indicated nutrients and contain-
ing 2% glucose to log phase. For mRNA decay analysis,
the yeast strain YTC345 carrying a temperature-sensitive
RNA polymerase II allele (rpb1-1) (28) was grown at 25C
in synthetic medium lacking the indicated nutrients and
containing 2% glucose until an OD
600
of 1.25 was at-
tained and then shifted to a 37C water bath shaker to
block transcription activity of RNA polymerase II.
Aliquots were collected at the indicated time points after
transcription shut-off for total RNA isolation and
northern blot analysis. Total RNA was prepared by the
hot acid phenol method (25) and 10 mg of each total RNA
sample was separated by 1.2% agarose gel electrophoresis
in the presence of 3.7% formaldehyde. Transfer to nylon
membrane (Millipore) was achieved by capillary action
with 20SSC. Blots were probed with
32
P-radiolabeled
riboprobes directed against the genes as indicated. The
level of mRNA in the northern blots was determined by
quantifying the intensity of bands using Image J software,
in three independent experiments, normalized against the
intensity of SCR1 RNA, which is a stable polymerase III
transcript (29), and graphed with Microsoft Excel. For
quantification of relative POR1 mRNA levels, the values
were set to 1 in wild-type yeast. For relative EDC1 mRNA
levels, the values were set to 1 in dhh1Dmutant yeast.
Mean values SD are shown.
Immunoprecipitation and POR1 mRNA detection by
RT–PCR
Exponentially growing cells (OD
600
10) were disrupted
with glass beads in 0.4 ml of extraction buffer [25 mM
HEPES–KOH, pH 7.5, 75 mM KCl, 2 mM MgCl
2
, 0.1%
NP-40, 1 mM DTT, 0.2 mg/ml heparin, 20 U/ml DNase
(TaKaRa) and 10 mg/ml aprotinin, leupeptin, and
pepstatin]. Extracts were cleared by centrifugation at
4000 g for 10 min. Monoclonal anti-HA antibody-
conjugated agarose beads (mouse monoclonal anti-HA-
Agarose antibody) (Sigma #A2095) was added to the
cleared extracts and incubated at 4C for 4 h. Beads
were washed four times with wash buffer (25 mM
HEPES–KOH, pH 7.5, 75 mM KCl, 2 mM MgCl
2
, 0.1%
NP-40) and the bound complexes were eluted with 50 mM
Tris–HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1% SDS
for 10 min at 65C. The RNA from cell extract (Input) and
immunoprecipitates (IP) were extracted by the hot acid
phenol method and used as template for RT–PCR.
POR1,COR1,orSED1 mRNA was detected by RT–
PCR with equal amounts of RNA from each sample
using a RvertAid H minus First Strand cDNA Synthesis
kit (Fermentas) following conditions suggested by the
manufacturer. The number of amplification cycles was
adjusted to avoid reaching a plateau phase during PCR.
One-third of each reaction was separated on a 1.5%
agarose gel and stained with ethidium bromide. The
RT–PCR products from precipitated POR1 mRNA were
quantified and normalized to RT–PCR products from
input mRNA by Image J software. Values represent the
mean of three independent experiments SD. Statistical
significance was assessed by the t-test (**P<0.001;
***P<0.0001). Endogenous Rbp1p or HA-tagged
proteins from cell extract and immunoprecipitate was
separated on a 9% SDS–PAGE gel, blotted and hybrid-
ized with anti-Dhh1p or anti-Rbp1p antibody for the
presence of proteins.
RESULTS
Dhh1p is involved in Rbp1p-mediated porin mRNA decay
We have previously described that Rbp1p specifically
binds to the 30-UTR of POR1 mRNA and promotes its
specific degradation in an Xrn1p-dependent pathway (25,
26). To explore the potential participation of decapping
activators Dhh1p, Pat1p, or Lsm1p in Rbp1p-mediated
Nucleic Acids Research, 2012, Vol. 40, No. 3 1333
POR1 mRNA decay, we overexpressed Rbp1p in wild-
type, dhh1D,pat1D, lsm1D, and xrn1Dmutant cells,
and examined their steady-state POR1 mRNA levels.
Northern blot analysis (Figure 1A) shows that
overexpression of Rbp1p in dhh1Dor xrn1Dmutant cells
had no effect on the level of POR1 mRNA, whereas the
POR1 mRNA level was reproducibly decreased in Rbp1p-
overexpressing pat1D,lsm1D, or wild-type cells. This result
suggests that Dhh1p, but not other decapping activators,
participates in Rbp1p-meidated POR1 mRNA decay.
To test if Rbp1p and Dhh1p could act on POR1 mRNA
decay in the same pathway, we examined the
POR1 mRNA level in dhh1Dand rbp1Ddhh1Dstrains.
Figure 1B shows a significant increase in the POR1
mRNA level in the dhh1Dstrain, but no additional
increase in rbp1Ddhh1Ddouble mutant cells, suggesting
that Rbp1p and Dhh1p may regulate POR1 mRNA decay
cooperatively.
Figure 1. Rbp1p requires Dhh1p to elicit porin mRNA decay. (A) The steady-state POR1 mRNA levels in dhh1D,pat1D,lsm1D, and xrn1Dmutant
cells overexpressing Rbp1p. BY4741 wild-type or indicated mutant strains transformed with pVT101U or pVT101U-HA-RBP1 plasmid were grown
to log phase. Total RNA of these cells was extracted and analyzed by northern blotting. (B) Steady-state levels of POR1 mRNA in wild-type, rbp1D,
dhh1D, and rbp1Ddhh1Dmutant cells. BY4741 strains were grown to log phase. Total RNA samples were isolated and analyzed by northern blot.
(C) Effect of Dhh1p overexpression on POR1 mRNA steady-state level. BY4741 wild-type strain were transformed with pVT101U plus YEplac181,
pVT101U plus YEplac181-HA-RBP1, pVT101U-DHH1 plus YEplac181, or pVT101U-DHH1 plus YEplac181-HA-RBP1 and grown to log phase.
Total RNA samples were isolated and analyzed by northern blot. Total proteins were precipitated by TCA and analyzed by western blotting.
(D)POR1 mRNA turnover in dhh1strain overexpressing Rbp1p. YTC345 wild-type or dhh1Dstrain transformed with either pVT101U, or
pVT101U-HA-RBP1 plasmid was grown to log phase at 25C and then shifted to 37C. Total RNA was extracted at each indicated time point
after temperature shift and analyzed. t
1/2
indicated the half-life of POR1 mRNA. Graphical representation of the POR1 mRNA decay kinetics is
shown. The levels of the mRNAs in (B–D) were quantitated as described in ‘Materials and Methods’ section. Mean values SD are shown.
1334 Nucleic Acids Research, 2012, Vol. 40, No. 3
We next examined whether overexpression of Dhh1p
can decrease the POR1 mRNA level. Figure 1C shows
that the POR1 mRNA level was affected by over-
expression of Rbp1p, but not Dhh1p, in wild-type cells.
In addition, the extent of POR1 mRNA decrease in
yeast co-expressing Rbp1p and Dhh1p is similar to that
in yeast overexpressing Rbp1p (Figure 1C). These results
indicate that Dhh1p alone is not sufficient to decrease
POR1 mRNA levels and suggest that Rbp1p plays
as a primary regulator for the degradation of POR1
mRNA.
We next examined whether the failure of overexpressing
Rbp1p to decrease the steady-state level of POR1 mRNA
in the dhh1Dstrain (Figure 1A) is due to its inability to
promote POR1 mRNA decay. We took advantage of an
RNA polymerase II temperature-sensitive mutant (rpb1-1)
strain (28), which allows transcriptional shutoff by tem-
perature shift, and evaluated the mRNA turnover rate in
the presence of overexpressed Rbp1p. The half-life of
POR1 mRNA in these cells was measured by northern
blotting. As shown in Figure 1D, the half-life of POR1
mRNA is prolonged in dhh1Dmutant cells (60 min) and
overexpression of Rbp1p in dhh1Dmutant cells had no
effect on the half-life of POR1 mRNA. The turnover of
SED1 mRNA, which encodes an abundant cell-wall
protein (30), served as a control, indicating that the
decay machinery was not impaired by DHH1 deletion.
The pronounced stabilization of POR1 mRNA in the
dhh1Dstrain overexpressing Rbp1p provides evidence
that Dhh1p participates in Rbp1p-mediated POR1
mRNA decay. Together, our data demonstrate that
Dhh1p is required for the efficiency of Rbp1p-mediated
POR1 mRNA decay.
Dhh1p-mediated EDC1 mRNA level is independent of
Rbp1p
Previous studies showed that DHH1, but not PAT1 and
LSM1, is involved in regulating the level of EDC1 mRNA
(15,31). Therefore, we examined whether Rbp1p is also
involved in regulation of EDC1 mRNA decay.
Consistent with previous reports (15,31), Figure 1A and
B show that the steady-state level of EDC1 mRNA is sig-
nificantly increased in the dhh1Dand xrn1Dstrains and the
half-life of EDC1 mRNA in the dhh1Dstrain is prolonged
for >60 min (Figure 1D). However, unlike POR1 mRNA,
the steady-state level and turnover rate of EDC1 mRNA
were not altered in cells overexpressing Rbp1p (Figure 1A
and D). In addition, the steady-state level of
EDC1 mRNA is not affected in rbp1Dmutant cells
(Figure 1B). These results indicate that Rbp1p is not
involved in Dhh1p-mediated down-regulation of EDC1
mRNA.
Growth impairment caused by RBP1 overexpression is
partially rescued in dhh1"mutant cells
Previous results have shown that overexpression of Rbp1p
impairs cell growth (24), however, overexpression of the
RNA binding-defective RBP1-RRM mutants had no such
effect. Therefore, cell growth defects caused by RBP1
overexpression might be a consequence of aberrant
biogenesis of specific mRNAs regulated by Rbp1p. To
reproduce these observations, we genetically manipulated
the expression of RBP1 under the control of a galactose-
inducible GAL1 promoter. As expected, no obvious differ-
ence in growth rate was observed between wild-type cells
expressing RBP1 by its own promoter or by GAL1
promoter when grown on glucose-containing medium;
whereas a significant growth defect was observed for
cells carrying GAL1 promoter-controlled Rbp1p when
grown in galactose-containing medium plates or liquid cul-
tures, as compared to the wild-type cells (Supplementary
Figure S1A and S1B). If Dhh1p is required for facilitating
specific Rbp1p-mediated mRNA decay, the impaired
growth caused by Rbp1p overexpression may be rescued
in dhh1mutant cells. We next assayed the effect of
Rbp1p overexpression on the growth rate of yeast
strains lacking Dhh1p. Although Dhh1p is involved in
Rbp1p-mediated POR1 mRNA decay, we found that de-
letion of DHH1 can only partially rescue cell growth im-
pairment caused by Rbp1p overexpression
(Supplementary Figure S1A and S1B). Quantification of
differential growth after 25-h incubation in liquid cultures
is shown in Supplementary Figure S1C. Furthermore,
using the same GAL1 promoter-controlled Rbp1p expres-
sion assay, we found that cell growth impairment caused
by overexpressing Rbp1p was not affected when PAT1 or
LSM1 gene was deleted (Supplementary Figure S1A).
These results suggest that Dhh1p, but not Pat1p and
Lsm1p, may play a partial role in the mechanism of
Rbp1p-mediated mRNA metabolism.
The non-conserved C-terminus of Dhh1p is required
for its interaction with Rbp1p
Our previous study has shown that Dhh1p interacted
with Rbp1p [(26) and data not shown]. We speculated
that Rbp1p could recruit Dhh1p to Rbp1p-specific
target mRNAs and then promote their degradation via
the general mRNA turnover pathway. Dhh1p consists of
two RecA-like domains, like all DEAD-box family
members and also contains amino- and carboxy-terminal
glutamine-rich extensions [(15) and Figure 2A]. Here, we
further narrowed down the interaction region of the Dhh1p
C-terminus using yeast two-hybrid assays. By a series of
C-terminal deletion mutants of Dhh1p (Figure 2A), and
showed that deletion of the C-terminal 81 amino acids
of Dhh1p, in the glutamine-rich region, completely abol-
ished the interaction of Dhh1p with Rbp1p (Figure 2B).
However, the C-terminal 81 residues of Dhh1p were not
sufficient for interaction with Rbp1p (data not shown).
To confirm these observations, we also performed in vitro
GST pull-down assays using purified recombinant GST-
Rbp1p-CF protein, which is a C-terminal fragment of
Rbp1p and interacts with Dhh1p (data not shown).
Figure 2C shows that purified His-tagged Dhh1p, but
not His-tagged Dhh1p-dC81, was efficiently pulled down
by GST-Rbp1p-CF. Together, these results indicate that
the C-terminal 81 residues mediate the Dhh1p–Rbp1p
interaction, which may allow the recruitment of Dhh1p
by Rbp1p to specific mRNAs.
Nucleic Acids Research, 2012, Vol. 40, No. 3 1335
The non-conserved C-terminus of Dhh1p is involved in
regulating Rbp1p-mediated POR1 mRNA decay,
but not the EDC1 mRNA level
To determine the significance of the in vitro direct inter-
action between Dhh1p and Rbp1p on POR1 and EDC1
mRNA decay, we examined the levels of these mRNAs in
dhh1Dmutant cells expressing full-length Dhh1p,
Dhh1p-dC81, or Dhh1p-dC106. We hypothesized that
Dhh1p lacking the C-terminal 81 residues would not
restore POR1 mRNA levels as sufficient as wild-type
protein caused by loss of its interaction with endogenous
Rbp1p. As shown in Figure 3A, the POR1 mRNA level in
the dhh1Dstrain expressing Dhh1p-dC81 was not fully
restored as compared with the dhh1Dstrain expressing
wild-type protein. Compared with the wild-type strain,
there was a slight increase in the level of POR1 mRNA
in the dhh1Dstrain carrying Dhh1p-dC81, which was com-
parable to the level observed in the rbp1Dstrain. In
contrast to POR1, the level of EDC1 mRNA in the
dhh1Dstrain was restored by expression of Dhh1p or
Dhh1p-dC81 (Figure 3A). These results indicate that
Dhh1p-dC81 failed to induce Rbp1p-mediated regulation
of POR1, but was capable of regulating the mRNA level
of EDC1. Dhh1p-dC106, lacking the entire non-conserved
C-terminal region and part of RecA-like domain II that
resulted in a loss of interaction with Rbp1p (Figure 2),
could restore the mRNA levels of neither POR1
nor EDC1 (Figure 3A). Western blots indicate that
this effect is not due to differences in protein expression
level (Figure 3A, lower panel). We further determined
whether the incomplete restoration of the POR1 mRNA
level in the dhh1Dstrain expressing Dhh1p-dC81 was
related to a failure to mediate POR1 mRNA decay. We
determined the POR1 mRNA turnover in an rpb1-1
temperature-sensitive mutant strain in which the DHH1
gene was deleted. We expressed single-copy DHH1 or
DHH1-dC81 in rpb1-1 dhh1Dstrain and analyzed
POR1 mRNA after shutoff transcription. Northern blots
(Figure 3B) show that the half-life of POR1 mRNA is
prolonged in dhh1Dmutant cells. The turnover rate of
POR1 mRNA was restored to normal (40 min) in
dhh1Dmutant cells expressing wild-type Dhh1p, but not
in the dhh1Dstrain carrying an empty vector or expressing
Dhh1p-dC81 (Figure 3B). Consistent with the steady-state
level (Figure 3A), the turnover rate of EDC1 mRNA in
the dhh1Dstrain expressing Dhh1p-dC81 was similar to
that in cells expressing Dhh1p (data not shown),
indicating that Dhh1p-dC81 does not affect the stability
of EDC1 mRNA. These results suggest that the insuffi-
cient restoration of POR1 mRNA degradation in dhh1D
mutant cells by Dhh1p-dC81 may be due the loss of inter-
action with Rbp1p. Taken together, these data indicate
that non-conserved C-terminal region of Dhh1p is not
critical for regulating EDC1 mRNA level, but is
required for Rbp1p-mediated POR1 mRNA decay.
The non-conserved C-terminus of Dhh1p is dispensable for
the conserved function and localization of Dhh1p
Although the DHH1 gene is not essential for yeast cell
viability, the dhh1Dstrain has been shown to display a
Figure 2. The non-conserved C-terminal 81 residues of Dhh1p are required for its interaction with Rbp1p. (A) Schematic representation of the
Dhh1p protein domain structure and C-terminal truncated variants used in yeast two hybrid and in vitro pull-down assays. (B) Dhh1p interacts with
Rbp1p through C-terminal 81 residues in yeast two-hybrid assay. YEM1acells expressing LexA- and Gal4AD-fusion proteins as indicated were used
to perform b-galactosidase reporter assay. Immunoblotting shows the expression level of indicated proteins. (C) Dhh1p directly binds Rbp1p. In vitro
pull-down assay between purified GST-tagged Rbp1p-CF and His-tagged Dhh1p or Dhh1p-dC81. Anti-His and anti-GST antibodies were used to
detect indicated fusion proteins in western blotting. The same amounts of fusion proteins in binding reaction were loaded as input controls.
1336 Nucleic Acids Research, 2012, Vol. 40, No. 3
temperature-sensitive growth phenotype (32). To examine
whether the non-conserved C-terminus of Dhh1p pos-
sesses other in vivo functions, we expressed C-terminal de-
letions of Dhh1p in the dhh1Dstrain and determined
the functional complement of the temperature-sensitive
phenotype. We observed that Dhh1p-dC81, like Dhh1p,
fully complemented the capacity of dhh1Dmutant cells to
grow at a non-permissive temperature (Figure 4A). In
contrast, expression of Dhh1p-dC106 failed to comple-
ment the growth defect in the dhh1Dstrain (Figure 4A).
Even though Dhh1p-dC106 was expressed from a
high-copy 2 mplasmid to increase protein levels similar
to wild-type Dhh1p (Figure 4B), the complementation of
the dhh1Dstrain to grow at the non-permissive tempera-
ture still failed (Figure 4A). These data indicate that the
non-conserved C-terminal 81 residues are not needed for
function of Dhh1p in cell proliferation.
Since Dhh1p is a component of cytoplasmic P-bodies
and accumulates with Dcp2p in P-bodies under different
cell stress conditions (33), we therefore asked whether the
non-conserved C-terminal 81 residues of Dhh1p are
required for its localization to P-bodies. We expressed
C-terminal GFP-fusion Dhh1p, Dhh1p-dC81, or Dhh1p-
dC106 in yeast with a P-bodies marker, Dcp2p-mCherry.
We examined P-body formation under glucose depriv-
ation, which leads to rapid localization of Dhh1p to
Dcp2p-marked P-bodies (33). As shown in Figure 4C,
GFP-fusion Dhh1p and Dhh1p-dC81 showed accumula-
tion to cytoplasmic foci and co-localization with the
P-bodies marker Dcp2p-mCh under stress. However, the
GFP-fusion Dhh1p-dC106 showed reduced accumulation
with the P-bodies marker Dcp2p-mCh under the same
treatment (Figure 4C). As the protein level of the
GFP-fusion Dhh1p-dC106 was equal to that of Dhh1p,
we ruled out that its reduced P-bodies localization is due
to lower protein levels (Figure 4D). These results indicate
the unique C-terminal domain of yeast Dhh1p was not
critical for its P-body localization.
Dhh1p associates with POR1 mRNA in vivo via its
interaction with Rbp1p
To determine whether Dhh1p can associate with POR1
mRNA in vivo, we precipitated Dhh1p-containing mRNP
complexes and detected the presence of POR1 mRNA by
RT–PCR. Cell extracts prepared from a strain carrying the
chromosomal DHH1 tagged with 3HA was used for
immunoprecipitation using anti-HA antibody-conjugated
beads. As shown in Figure 5A, anti-HA beads efficiently
precipitated the Dhh1p–3HA protein from yeast extracts
and endogenous POR1 mRNA was detected from
immunoprecipitates of Dhh1p–3HA (Figure 5A).
We further examined whether POR1 mRNA associ-
ation with Dhh1p is dependent on the presence of
Rbp1p. To address this, we immunoprecipitated Dhh1p–
3HA protein complexes from an rbp1Dstrain and analyzed
the presence of POR1 mRNA. In contrast to the DHH1-
3HA wild-type strain, we did not detect POR1 mRNA
from Dhh1p–3HA precipitates in the rbp1Dstrain
(Figure 5A). The PCR product was not detected when
reverse transcriptase was omitted from the input fraction,
indicating that formation of bands in the immunopre-
cipitate fraction is from cDNA. The interaction of
Dhh1p with endogenous Rbp1p was confirmed in
Dhh1p–3HA precipitates by immunoblotting using anti-
Rbp1p antibody (Figure 5A). Our observations indicate
Figure 3. The C-terminal 81 residues of Dhh1p are required for
Rbp1p-mediated POR1 mRNA decay. (A) Steady-state POR1 mRNA
levels in dhh1Dstrain expressing Dhh1p, Dhh1p-dC81, or
Dhh1p-dC106. BY4741dhh1Dstrain carrying YCplac111, YCplac111-
DHH1, YCplac111-DHH1-dC81, or YCplac111-DHH1-dC106 was
grown to log phase and total RNA were extracted and analyzed by
northern blotting. Total proteins were precipitated by TCA and
analyzed by western blotting. (B) Turnover of POR1 mRNA in
dhh1Dstrain expressing Dhh1p or Dhh1p-dC81. YTC345dhh1Dstrain
carrying YCplac111, YCplac111-DHH1, or YCplac111-DHH1-dC81
was grown to log phase at 25C and then shifted to 37C. Total
RNA was extracted at each indicated time point after temperature
shift and analyzed. t
1/2
indicated the half-life of POR1 mRNA.
Graphical representation of the POR1 mRNA decay kinetics is
shown. The levels of the mRNAs were quantitated as described in
‘Materials and Methods’ section. Mean values SD are shown.
Nucleic Acids Research, 2012, Vol. 40, No. 3 1337
that Dhh1p can in vivo associate with POR1 mRNA and
Rbp1p, and suggest that the association of POR1 mRNA
to Dhh1p might be through Rbp1p.
It has been suggested that the target RNA specificity
of DEAD-box proteins can be determined by their inter-
acting partners (14,16,17). The above observations
indicate that the association of the POR1 mRNA with
Dhh1p was dependent on its interacting partner Rbp1p.
Therefore, it is possible that the C-terminal Rbp1p inter-
acting region of Dhh1p is required for its association with
POR1 mRNA. To verify this possibility, we precipitated
Dhh1p–3HA or Dhh1p-dC81-3HA protein from DHH1-
3HA and DHH1-dC81-3HA strains. Using RT–PCR, we
detected POR1 mRNA from the precipitates of DHH1-
3HA but not from that of DHH1-dC81-3HA (Figure 6B).
Endogenous Rbp1p was only detected in the Dhh1p–3HA
protein complex but not the Dhh1p-dC81-3HA complex
(Figure 5B). From these data, we propose that Dhh1p
associates with POR1 mRNA via its interaction with
Rbp1p.
To further support this hypothesis, we proposed that
the inability of the POR1 mRNA to associate with
Dhh1p in the rbp1Dstrain could be rescued by wild-type
Rbp1p, but not the mutants of Rbp1p lacking the ability
of interacting with Dhh1p or binding to POR1 mRNA.
Two Rbp1p mutants were used in this test; Rbp1p-dNMP,
a C-terminally deleted Rbp1p, which lost interaction with
Dhh1p (Supplementary Figure S2A), and Rbp1p-rrm1,
which is defective for POR1 mRNA-binding activity
but still possesses ability to interact with Dhh1p [(25)
and Supplementary Figure S2B]. We analyzed POR1
mRNA from Dhh1p protein complexes in rbp1Dstrains
expressing exogenous wild-type Rbp1p, Rbp1p-dNMP or
Rbp1p-rrm1. As shown in Figure 5C, Rbp1p, but not
Rbp1p-dNMP, was co-precipitated with Dhh1p and
could associate with POR1 mRNA in the rbp1Dstrain.
Figure 4. Dhh1p-dC81 complements the temperature-sensitive phonotype of dhh1Dstrain and efficiently accumulates to P-bodies. (A) Dhh1p-dC81
complements the growth defect of dhh1Dmutant strain. BY4741 wild-type or dhh1Dstrain expressing Dhh1p, Dhh1p-dC81, or Dhh1p-dC106 from
CEN or 2 mplasmid was grown to log phase, serially diluted and spotted on two plates, which were then separately incubated at 30Cor37
C for 2
days. (B) Isometric expression level of various Dhh1p mutants. Immunoblotting confirmed the protein expression of various Dhh1p mutants in panel
A. (C) Dhh1p-dC81-GFP localizes to P-bodies under glucose deprivation. BY4741 wild-type cells chromosomally expressing C-terminal tagged
GFP-fusion Dhh1p, Dhh1p-dC81 or Dhh1p-dC106 with the P-bodies marker Dcp2p-mCherry were grown in YPD medium to log phase and then
shifted to medium lacking glucose for 20 min. Co-localization of fluorescence fusion proteins was quantitated as described in ‘Materials and Methods’
section (D) Isometric expression level of various GFP-fusion Dhh1p mutants. Immunoblotting confirms the protein expression of GFP-fusion
Dhh1p, Dhh1p-dC81 and Dhh1p-dC106.
1338 Nucleic Acids Research, 2012, Vol. 40, No. 3
This result supports the notion that interaction with
Rbp1p is required for Dhh1p association with POR1
mRNA. Another mutant, Rbp1p-rrm1, also failed to
restore the association of POR1 mRNA with Dhh1p in
the rbp1pDstrain, although it could interact with Dhh1p
in vivo (Figure 5C). Furthermore, in the absence of Rbp1p,
Dhh1p–3HA could associate in vivo with one of the
known Dhh1p association mRNAs, COR1 mRNA (34),
but not the control SED1 mRNA, whose expression is not
sensitive to the dhh1Dmutant (Figure 1D) and is not
immunoprecipitated by Dhh1p–3HA (Figure 5A–C).
These results indicate that our immunoprecipitation
assay could detect specific in vivo Dhh1p–mRNP
complexes. Together, these results indicate that proper
POR1 mRNA association with Dhh1p not only required
Dhh1p interaction with Rbp1p but also depended on the
RNA-binding ability of Rbp1p.
To test if Rbp1p binding to POR1 mRNA requires
Dhh1p, we fused three HA tags at the C-terminus of
endogenous Rbp1p in dhh1Dor wild-type strains. Yeast
extracts were prepared and precipitated as described
above. POR1 mRNA could be detected in Rbp1p–3HA
precipitates from both wild-type and dhh1Dstrains
(Figure 5D), indicating a Dhh1p-independent association
of Rbp1p and POR1 mRNA in vivo. In contrast to POR1
mRNA, EDC1 mRNA was not precipitated from the
Rbp1p–3HA protein complex even though its level highly
increased in the absence of Dhh1p. We also identified that
Dhh1p was co-precipitated from the Rbp1p protein
complex (Figure 5D), indicating that POR1 mRNA, but
not EDC1 mRNA, specifically associates with the Rbp1p–
Dhh1p protein complex.
Mammalian DDX6 fused with the C-terminal
non-conserved region of Dhh1p confers Rbp1p-mediated
porin mRNA decay in dhh1Dmutant cells
The Dhh1p protein and its homologs of different species
are highly conserved in function, because Xenopus Xp54
Figure 5. Dhh1p associated with POR1 mRNA in vivo requires its interaction with Rbp1p. (A) Dhh1p association with POR1 mRNA depends on
the presence of Rbp1p. Dhh1p chromosomally tagged with three HA epitopes in wild-type or rbp1Dstrain was immunoprecipitated and used to
perform RT–PCR as described in ‘Materials and Methods’ section. (B) Dhh1p associates with POR1 mRNA through C-terminal 81 amino acids.
Dhh1p or Dhh1-dC81 chromosomally tagged with three HA epitopes was immunoprecipitated and used to perform RT–PCR as described in
‘Materials and Methods’ section. (C) Dhh1p association with POR1 mRNA in rbp1Dmutant was rescued by Rbp1p, but not Rbp1p-dNMP or
Rbp1p-rrm1. DHH1-3HA rbp1Dstrains carrying pVT101U, pVT101U-RBP1, pVT101U-RBP1-dNMP, or pVT101U-RBP1-rrm1 were grown and
then used to perform immunoprecipitation and RT–PCR as described in ‘Materials and Methods’ section. (D) Rbp1p specifically associates with
POR1 mRNA independent of Dhh1p. Rbp1p chromosomally tagged with three HA epitopes in wild-type or dhh1Dstrain was immunoprecipitated
and used to perform RT–PCR as described in ‘Materials and Methods’ section. The presence of Dhh1p or Rbp1p was shown in western blots. The
level of the RT–PCR products of POR1 mRNA was quantitated as described in ‘Materials and Methods’ section. Mean values SD are shown.
Nucleic Acids Research, 2012, Vol. 40, No. 3 1339
Figure 6. Mammalian DDX6 fused with the non-conserved C-terminal 85 residues of Dhh1p gains function to confer specific regulation for
Rbp1p-mediated porin mRNA. (A) Schematic representation of the Dhh1p, DDX6 and DDX6 chimera protein domain structure. Portion of
DDX6 chimera protein, DDX6-C85, derived from Dhh1p are shown. (B) DDX6 and its chimera protein can complement the growth defect of
dhh1Dstrain. BY4741dhh1Dstrain expressing Dhh1p, DDX6 or DDX6-C85 was used to perform growth assays as described in Figure 4A. Protein
expression level was shown by western blotting. (C) Steady-state POR1 mRNA levels in dhh1Dstrain expressing DDX6 or DDX6-C85.
BY4741dhh1Dstrain expressing DDX6 or DDX6-C85 from 2-mplasmid was grown to log phase and total RNA were extracted and analyzed.
The levels of the mRNAs were quantitated as described in ‘Materials and Methods’ section. (D) DDX6 chimera protein DDX6-C85, but not DDX6,
interacts with Rbp1p in yeast two-hybrid assay. YEM1acells expressing LexA- and Gal4AD-fusion proteins as indicated were used to perform
b-galactosidase reporter assays. Immunoblotting shows the expression level of indicated proteins. (E) DDX6-C85 associates with POR1 mRNA
in vivo through interaction with Rbp1p. BY4741dhh1Dstrain expressing Dhh1p-2HA, DDX6-2HA or DDX6-C85-2HA were used to perform
immunoprecipitation and RT–PCR as described in ‘Materials and Methods’ section. The level of RT–PCR products of POR1 mRNA was
quantitated as described in ‘Materials and Methods’ section.
1340 Nucleic Acids Research, 2012, Vol. 40, No. 3
(32) and mammalian RCK/p54/DDX6 (35–37) could
complement the growth defects of a yeast dhh1Dmutant.
We therefore asked if human DDX6 is capable of
substituting for Dhh1p in mediating POR1 mRNA
decay in yeast cells. As shown in Figure 6C, the POR1
mRNA level was not fully restored in the dhh1Dstrain
expressing human DDX6. This result was similar to the
effect of POR1 mRNA level in the dhh1Dstrain expressing
Dhh1p-dC81 (Figure 3A). Although the non-conserved
C-terminal region of Dhh1p was not sufficient to interact
with Rbp1p, we proposed that it contains a critical
element to determine the specific interacting structure for
Dhh1p. We further tested whether the human DDX6
fused with the non-conserved C-terminus of Dhh1p can
confer Rbp1p-mediated POR1 mRNA decay in dhh1D
strain. We generated a DDX6-Dhh1p(C-terminus) fused
protein, DDX6-C85(Dhh1p), containing the C-terminal
85 residues of Dhh1p appended to the C-terminus of
DDX6, as illustrated in Figure 6A. Northern blotting
shows that, in contrast to DDX6, DDX6-C85(Dhh1p)
can restore the level of POR1 mRNA in dhh1Dstrain
(Figure 6C). Interestingly, the level of EDC1 mRNA in
the dhh1Dstrain was restored by DDX6 and DDX6-
C85(Dhh1p) although at varied degrees (Figure 6C), sug-
gesting that a certain specific motif in Dhh1p might be
important to regulate the EDC1 mRNA level. These
data indicate that DDX6 fused with the non-conserved
C-terminus of Dhh1p could complement the function of
Dhh1p in dhh1Dmutant cells. We also confirmed prior
reports for phenotype complementation (35–37) that
DDX6 and DDX6-C85(Dhh1p) restored the capacity of
dhh1Dstrain to grow at a non-permissive temperature
(Figure 6B).
The ability of DDX6-C85(Dhh1p) to mediate porin
mRNA decay suggested that it might possess the ability
to interact with Rbp1p and subsequently to associate with
POR1 mRNA in vivo. To test this hypothesis, we assayed
the interaction between Rbp1p and DDX6-C85(Dhh1p)
using the yeast two-hybrid assay. Figure 6D shows that
DDX6-C85(Dhh1p) could interact with Rbp1p. We
further examined what besides the C-terminal region is
sufficient for the Rbp1p interaction. Based on the struc-
tural regions of Dhh1p (15,16), we constructed two
deletion mutants of Dhh1p, Dhh1p-VI and Dhh1p-Ct,
which contain motif VI or RecA-like domain II,
adjacent to non-conserved C-terminal region, respectively
(Supplementary Figure S3A). We found that Dhh1p-Ct,
but not Dhh1p-VI, had similar interaction intensity as
full-length Dhh1p (Supplementary Figure S3B). We also
demonstrated that DDX6-Ct-C85, which contains RecA-
like domain II fused with non-conserved C-terminal
region of Dhh1p, but not DDX6-Ct, is sufficient for the
Rbp1p interaction. This result indicates that the RecA-like
domain II contributes to the interaction between Dhh1p
and Rbp1p. Next, we performed RNA immunopre-
cipitation in the dhh1Dstrain expressing Dhh1p-2HA,
human DDX6-2HA or DDX6-C85(Dhh1p)-2HA. As
shown in Figure 6E, we detected POR1 mRNA from
Dhh1p–2HA and DDX6-C85(Dhh1p)-2HA, but not
from DDX6–2HA precipitates. Immunoblotting shows
that endogenous Rbp1p can be detected in Dhh1p–2HA
and DDX6-C85(Dhh1p)–2HA, but not DDX6–2HA pre-
cipitates (Figure 6E). Furthermore, the interaction be-
tween Dhh1p or DDX6-C85 and endogenous Rbp1p
was insensitive to RNase treatment (Supplementary
Figure S4), indicating that the interaction is not due to
RNA bridging. These results suggest that DDX6-
C85(Dhh1p), but not DDX6, can mediate the POR1
mRNA decay through interaction with endogenous
Rbp1p via its fusion with the non-conserved C-terminus
of Dhh1p.
DISCUSSION
In this report, we show that the non-conserved C-terminus
of Dhh1p has a role in defining specific interactions with
Rbp1p and promotes POR1, but not EDC1 mRNA decay.
Moreover, mammalian DDX6, after being fused with the
non-conserved C-terminus of Dhh1p, possesses the ability
to interact with Rbp1p and to confer Rbp1p-mediated
POR1 mRNA decay in a dhh1strain. We propose that
promoting a distinct mRNA decay by Dhh1p may be
defined by specific RNA-binding proteins and/or regulat-
ing factors that interact with its non-conserved extended
C-terminus.
Dhh1p participates in Rbp1p-mediated POR1 mRNA
decay
By analyzing the POR1 mRNA level in deletion mutants,
we found that the magnitude of the effect of DHH1-
deletion, but not PAT1-orLSM1-deletion, on the
POR1 mRNA level was comparable to that of XRN1-
deletion. In addition, only in dhh1D, but not pat1Dor
lsm1Dmutant cells, overexpression of Rbp1p failed to de-
stabilize POR1 mRNA. Dhh1p physically interacts with
Pat1p (7). Although both proteins carry out similar func-
tions, they now appear to operate through separate mech-
anisms, which are not known in detail (16). Consistent
with this, our data show that POR1 mRNA levels are
not affected in PAT1-deleted yeast. Interestingly,
although both dhh1Dand rbp1Dstrains showed delays in
the POR1 mRNA decay rate, only the dhh1Dstrain ex-
hibited a corresponding large increase in steady-state
POR1 mRNA levels. This discrepancy between decay
rates and steady-state levels is similar to what has been
reported for EDC1 mRNA in the dhh1Dstrain and some
mRNAs in dcp1Dstrains (31,38). This result suggests that
there might be Dhh1p-dependent and Rbp1p-independent
pathways for POR1 mRNA degradation. It also implies
that other mRNA-binding regulator(s) can cooperate
with Dhh1p to regulate POR1 mRNA degradation.
Furthermore, overexpression of Dhh1p does not lead to
destabilization of POR1 mRNA. This indicates that
Dhh1p is not the limiting factor in POR1 mRNA decay,
and that Dhh1p requires Rbp1p or other RNA-binding
molecules to promote POR1 mRNA degradation.
In agreement with a previous report (31), we observed a
large increase in EDC1 mRNA levels in the dhh1Dstrain.
Our preliminary data showed that the decrease in RNA
decay efficiency of both POR1 and EDC1 mRNA in the
dhh1Dstrain was rescued by wild-type, but not helicase
Nucleic Acids Research, 2012, Vol. 40, No. 3 1341
mutants of Dhh1p, signifying the importance of Dhh1p
helicase activity in the degradation of both mRNAs.
However, unlike POR1 mRNA, the level of EDC1
mRNA was not affected by Rbp1p. Moreover, deletion
of DHH1 can only partially rescue cell growth impairment
caused by Rbp1p overexpression, suggesting that Dhh1p
only plays a partial role in Rbp1p-mediated mRNA me-
tabolism. Together, we believe that Rbp1p is not a univer-
sal partner for the Dhh1p-dependent mRNA degradation
pathway and that Dhh1p might modulate the decay of
different mRNAs through cooperating with different
molecules.
The non-conserved C-terminus of Dhh1p is involved in
Rbp1p-mediated POR1 mRNA
The extended C-terminal non-conserved region of Dhh1p
has been proposed to contribute to protein–protein inter-
actions and confer substrate specificity, thereby facilitat-
ing unique functions (13,14,16). In this report, we found
that the C-terminal non-conserved region is involved in
Rbp1p-mediated POR1 mRNA and is necessary for its
interaction with Rbp1p. Previous study has shown that a
Dhh1p deleted C-terminal 81 residues still conforms to a
typical helicase structure and retains its helicase-
dependent function (15), suggesting that the interaction
loss of Dhh1p-dC81 with Rbp1p was not due to disrup-
tion of overall protein conformation.
An interesting issue is the diversity of interactions that
may allow mRNA specific-binding proteins to recruit the
mRNA degradation machinery. Several studies have
shown that some mRNA-specific regulatory proteins
recruit the general repression and decay machinery to
specific transcripts, leading to transcript destabilization.
Yeast Cth2p was shown to interact with the C-terminal
catalytic core domain and non-conserved region of Dhh1p
and recruit it to ARE-containing mRNAs to promote
mRNA decay (39). The similar region of Dhh1p also
physically interacts with the Dcp1p–Dcp2p decapping
complex and the Edc3p scaffold protein, thereby pro-
moting the transition of mRNAs from translation to
decapping and 50–30degradation at specific intracellular
sites known as processing bodies (P-bodies) (40). Mpt5p
was reported to interact with Pop2p, thereby inducing the
recruitment of the Ccr4p, Dhh1p and Dcp2p to specific
mRNAs to promote mRNA deadenylation and decay
(41). Our data, consistent with previous reports, show
that the recruitment of Dhh1p to Rbp1p is mediated by
its non-conserved C-terminus, thereby promoting the
Rbp1p-mediated POR1 mRNA decay.
The non-conserved C-terminus of Dhh1p is dispensable
for conserved function of Dhh1p
Cell growth analyses and RNA decay assays show that
Dhh1p-dC81 is sufficient to complement the growth
defect and restore EDC1 mRNA degradation in a dhh1D
mutant strain. This observation is parallel to previous
reports that the vertebrate orthologs Xenopus Xp54 (32)
and mammalian RCK/p54/DDX6 (36), which do not pos-
sess a C-terminal extension like Dhh1p, can complement
the growth defect in a dhh1Dmutant strain. Furthermore,
expression of DDX6 in a dhh1Dmutant strain can partial-
ly restore the efficiency of degradation of EDC1 mRNA.
Therefore, we suggest that the non-conserved C-terminal
region of Dhh1p is not required for known conserved
DEAD-box helicase Dhh1p function.
The non-conserved C-terminal region of Dhh1p is a Q/
N-rich stretch. Some mRNA-specific regulatory proteins
that recruit the translation repression and 50to 30decay
machinery and localize to cytoplasmic P-bodies also con-
tain similar Q/N-rich sequences. These Q/N-rich regions
within those proteins have been reported to be both
required and sufficient for protein aggregation into foci
under certain stress conditions (19). However, Reijns
et al. (19) found that GFP-tagged C-terminal non-
conserved region of Dhh1p do not aggregate under
stresses and GFP-tagged Dhh1p-dC (1–427) could still ac-
cumulate to the P-bodies. Among orthologs of Dhh1p,
neither Drosophila Me31B nor Caenorhabditis CGH1
contain such Q/N-rich sequences at their C-termini.
Nevertheless, these proteins could localize to granule-like
subcellular structures similar to P-bodies (21,22).
Compatibly, our data demonstrate that the C-terminal
non-conserved Q/N-rich region of Dhh1p is not respon-
sible for accumulation into P-bodies under stress
conditions.
Dhh1p–Rbp1p interaction is required for the association
of POR1 mRNA with Dhh1p
DEAD-box RNA helicase proteins are believed to modu-
late the structure of RNAs and ribonucleoprotein com-
plexes by disrupting RNA helices and RNA–protein
interactions. In addition to duplex unwinding, RNA
helicases display an array of additional activities. Most
prominently, several RNA helicases have been directly
shown to displace other proteins from RNA in an
active, ATP-dependent fashion (42). Protein displacement
or RNP remodeling is thought to be central to the physio-
logical function of RNA helicases, because RNAs are gen-
erally bound to other proteins in vivo (10). DEAD-box
helicase proteins generally possess in vitro RNA-binding
ability (15), however, in vivo specific RNA substrates are
not known for most of the DEAD-box helicase proteins
(11). Hogan et al. (34) have systematically searched for
RNAs directly associated with purified putative RNA-
binding proteins. Less than 10 RNAs were identified to
be associated directly with Dhh1p. This lack of RNA spe-
cificity in vitro may reflect the fact that the biologically
relevant substrate is an RNP, a rare RNA conformation,
or an RNA that is recognized only in the context of a
larger complex (43).
We previously have shown that Rbp1p exhibits obvious
characteristics of known RNA-binding motifs, RRMs
and mutation of conserved residues in these motifs result
in defective POR1 mRNA binding (25). Analysis from
immunoprecipitation of RNP complexes showed that
Dhh1p association with POR1 mRNA in vivo requires its
interaction with Rbp1p. In an rbp1Dstrain or cells ex-
pressing only RNA-binding deficient Rbp1p, Dhh1p
failed to associate with POR1 mRNA. This result indi-
cates that Dhh1p may not be able to form a stable
1342 Nucleic Acids Research, 2012, Vol. 40, No. 3
complex with mRNAs in vivo by itself. Our data support
the notion that proper POR1 mRNA association with
Dhh1p not only requires Dhh1p interaction with Rbp1p
but also depends on the RNA-binding ability of Rbp1p.
Highly conserved DEAD-box domain in Dhh1p/RCK/
Xp54/DDX6 mediates non-conserved C-terminus of Dhh1p
to modulate transcript-specific decay
Dhh1p belongs to a highly conserved DEAD-box helicase
subfamily that includes human RCK/p54/DDX6,
Xenopus Xp54, Drosophila Me31B and Caenorhabditis
CGH-1 (16). These proteins share high sequence homology
except at the extended N- and C-termini flanking the
conserved catalytic domains (16). The non-conserved
C-terminal region of Dhh1p is a unique extension sequence
in yeast. In contrast, the mammalian RCK/p54/DDX6
has a long N-terminal extension and only a short
C-terminal region.
Through expressing DDX6 in a dhh1Dstrain, we found
that in addition to complementing the growth defect of the
dhh1Dstrain, DDX6 also partially promoted the efficiency
of degradation of EDC1 mRNA; whereas, it lacks the
ability to interact with Rbp1p and elicit Rbp1p-mediated
POR1 mRNA degradation. Interestingly, we found that
the addition of the C-terminal non-conserved region of
Dhh1p to DDX6 is sufficient to interact with Rbp1p
and introduce POR1 mRNA as a target in yeast.
Because the RecA-like domain II of DDX6 fused with
the non-conserved C-terminal region of Dhh1p can inter-
act with Rbp1p, we reason that the evolutionarily con-
served helicase domain may assist the C-terminal region
of Dhh1p to interact with other factors, thereby
modulating transcript-specific decay.
In S. cerevisiae, a number of mechanisms exist by which
the decapping machinery can be selectively recruited to
specific mRNA substrates (31,39,44,45). It is interesting
to note that the non-conserved C-terminal domain of
Dhh1p may be responsible for the interaction with
Dcp2p, Edc3p, Cth2p and Rbp1p, which raises the
question of whether these interactions can be regulated
by cellular signaling. Consistent with this notion, our pre-
liminary data show that Rbp1p is phosphorylated at more
than 10 residues. It would also be interesting to elucidate
which phosphorylation sites on Rbp1p function as mRNA
decay activation domains.
Orthologs of Dhh1p in various organisms have been
shown to carry out a diverse array of cellular processes
while functional conservation has also been observed. Our
findings suggest that the unique C-terminal extension of
Dhh1p facilitates a species-specific function in yeast.
Whether other non-conserved N- and C-terminal exten-
sions of each ortholog could interact with certain
RNA-binding proteins to carry out more species-specific
functions and when these specific functions emerge or
diminish during evolution would be an interesting future
subject. On the other hand, some mRNA specific-binding
proteins may be responsible for conserved functions of
Dhh1p and DDX6, recruiting them to mRNAs such as
EDC1. In conclusion, the data presented in this report
elucidate the role of the species-specific non-conserved
C-terminus of Dhh1p in defining specific interaction
with an RNA-binding protein, Rbp1p, to promote
POR1 mRNA decay. The challenge for future studies
will be to understand how mRNA-specific regulators
recruit Dhh1p and coordinate multiple mechanisms of
post-transcriptional regulation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Tables I–II, Supplementary Figures 1–4.
ACKNOWLEDGEMENTS
The authors thank Drs Woan-Yuh Tarn, Tien-Hsien
Chang, Randy Haun and Chun-Fang Huang for critical
reading of the manuscript.
FUNDING
Funding for open access charge: National Health
Research Institutes, Taiwan, R.O.C. (NHRI-EX94-
9222BI, NHRI-EX96-9513SI) and Yung-Shin
Biomedical Research Funds (to F.-J.S.).
Conflict of interest statement. None declared.
REFERENCES
1. Wilusz,C.J. and Wilusz,J. (2004) Bringing the role of mRNA
decay in the control of gene expression into focus. Trends Genet.,
20, 491–497.
2. Parker,R. and Song,H. (2004) The enzymes and control of
eukaryotic mRNA turnover. Nat. Struct. Mol. Biol.,11, 121–127.
3. Garneau,N.L., Wilusz,J. and Wilusz,C.J. (2007) The highways
and byways of mRNA decay. Nat. Rev. Mol. Cell. Biol.,8,
113–126.
4. Coller,J. and Parker,R. (2004) Eukaryotic mRNA decapping.
Annu. Rev. Biochem.,73, 861–890.
5. Franks,T.M. and Lykke-Andersen,J. (2008) The control of
mRNA decapping and P-body formation. Mol. Cell.,32, 605–615.
6. Tharun,S., He,W., Mayes,A.E., Lennertz,P., Beggs,J.D. and
Parker,R. (2000) Yeast Sm-like proteins function in mRNA
decapping and decay. Nature,404, 515–518.
7. Coller,J.M., Tucker,M., Sheth,U., Valencia-Sanchez,M.A. and
Parker,R. (2001) The DEAD box helicase, Dhh1p, functions in
mRNA decapping and interacts with both the decapping and
deadenylase complexes. RNA,7, 1717–1727.
8. Fischer,N. and Weis,K. (2002) The DEAD box protein Dhh1
stimulates the decapping enzyme Dcp1. EMBO J.,21, 2788–2797.
9. Coller,J. and Parker,R. (2005) General translational repression by
activators of mRNA decapping. Cell,122, 875–886.
10. Linder,P. (2006) Dead-box proteins: a family affair–active and
passive players in RNP-remodeling. Nucleic Acids Res.,34,
4168–4180.
11. Rocak,S. and Linder,P. (2004) DEAD-box proteins: the driving
forces behind RNA metabolism. Nat. Rev. Mol. Cell. Biol.,5,
232–241.
12. Silverman,E., Edwalds-Gilbert,G. and Lin,R.J. (2003) DExD/
H-box proteins and their partners: helping RNA helicases
unwind. Gene,312, 1–16.
13. Cordin,O., Banroques,J., Tanner,N.K. and Linder,P. (2006) The
DEAD-box protein family of RNA helicases. Gene,367, 17–37.
14. Jankowsky,E. (2011) RNA helicases at work: binding and
rearranging. Trends Biochem. Sci.,36, 19–29.
Nucleic Acids Research, 2012, Vol. 40, No. 3 1343
15. Cheng,Z., Coller,J., Parker,R. and Song,H. (2005) Crystal
structure and functional analysis of DEAD-box protein Dhh1p.
RNA,11, 1258–1270.
16. Weston,A. and Sommerville,J. (2006) Xp54 and related
(DDX6-like) RNA helicases: roles in messenger RNP assembly,
translation regulation and RNA degradation. Nucleic Acids Res.,
34, 3082–3094.
17. Fairman-Williams,M.E., Guenther,U.P. and Jankowsky,E. (2010)
SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol.,
20, 313–324.
18. Sheth,U. and Parker,R. (2003) Decapping and decay of messenger
RNA occur in cytoplasmic processing bodies. Science,300,
805–808.
19. Reijns,M.A., Alexander,R.D., Spiller,M.P. and Beggs,J.D. (2008)
A role for Q/N-rich aggregation-prone regions in P-body
localization. J. Cell Sci.,121, 2463–2472.
20. Minshall,N., Thom,G. and Standart,N. (2001) A conserved role
of a DEAD box helicase in mRNA masking. RNA,7, 1728–1742.
21. Nakamura,A., Amikura,R., Hanyu,K. and Kobayashi,S. (2001)
Me31B silences translation of oocyte-localizing RNAs through
the formation of cytoplasmic RNP complex during Drosophila
oogenesis. Development,128, 3233–3242.
22. Navarro,R.E., Shim,E.Y., Kohara,Y., Singson,A. and
Blackwell,T.K. (2001) cgh-1, a conserved predicted RNA helicase
required for gametogenesis and protection from physiological
germline apoptosis in C. elegans. Development,128, 3221–3232.
23. Smillie,D.A. and Sommerville,J. (2002) RNA helicase p54
(DDX6) is a shuttling protein involved in nuclear assembly of
stored mRNP particles. J. Cell Sci.,115, 395–407.
24. Lee,F.J. and Moss,J. (1993) An RNA-binding protein gene
(RBP1) of Saccharomyces cerevisiae encodes a putative
glucose-repressible protein containing two RNA recognition
motifs. J. Biol. Chem.,268, 15080–15087.
25. Buu,L.M., Jang,L.T. and Lee,F.J. (2004) The yeast RNA-binding
protein Rbp1p modifies the stability of mitochondrial porin
mRNA. J. Biol. Chem.,279, 453–462.
26. Jang,L.T., Buu,L.M. and Lee,F.J. (2006) Determinants of Rbp1p
localization in specific cytoplasmic mRNA-processing foci,
P-bodies. J. Biol. Chem.,281, 29379–29390.
27. Longtine,M.S., McKenzie,A. 3rd, Demarini,D.J., Shah,N.G.,
Wach,A., Brachat,A., Philippsen,P. and Pringle,J.R. (1998)
Additional modules for versatile and economical PCR-based gene
deletion and modification in Saccharomyces cerevisiae. Yeast,14,
953–961.
28. Muhlrad,D., Decker,C.J. and Parker,R. (1995) Turnover
mechanisms of the stable yeast PGK1 mRNA. Mol. Cell. Biol.,
15, 2145–2156.
29. Felici,F., Cesareni,G. and Hughes,J.M. (1989) The most abundant
small cytoplasmic RNA of Saccharomyces cerevisiae has an
important function required for normal cell growth. Mol. Cell.
Biol.,9, 3260–3268.
30. Shimoi,H., Kitagaki,H., Ohmori,H., Iimura,Y. and Ito,K. (1998)
Sed1p is a major cell wall protein of Saccharomyces cerevisiae in
the stationary phase and is involved in lytic enzyme resistance.
J. Bacteriol.,180, 3381–3387.
31. Muhlrad,D. and Parker,R. (2005) The yeast EDC1 mRNA
undergoes deadenylation-independent decapping stimulated by
Not2p, Not4p, and Not5p. EMBO J.,24, 1033–1045.
32. Tseng-Rogenski,S.S., Chong,J.L., Thomas,C.B., Enomoto,S.,
Berman,J. and Chang,T.H. (2003) Functional conservation of
Dhh1p, a cytoplasmic DExD/H-box protein present in large
complexes. Nucleic Acids Res.,31, 4995–5002.
33. Teixeira,D., Sheth,U., Valencia-Sanchez,M.A., Brengues,M. and
Parker,R. (2005) Processing bodies require RNA for assembly
and contain nontranslating mRNAs. RNA,11, 371–382.
34. Hogan,D.J., Riordan,D.P., Gerber,A.P., Herschlag,D. and
Brown,P.O. (2008) Diverse RNA-binding proteins interact with
functionally related sets of RNAs, suggesting an extensive
regulatory system. PLoS Biol.,6, e255.
35. Alves-Rodrigues,I., Mas,A. and Diez,J. (2007) Xenopus Xp54 and
human RCK/p54 helicases functionally replace yeast Dhh1p in
brome mosaic virus RNA replication. J. Virol.,81, 4378–4380.
36. Westmoreland,T.J., Olson,J.A., Saito,W.Y., Huper,G., Marks,J.R.
and Bennett,C.B. (2003) Dhh1 regulates the G1/S-checkpoint
following DNA damage or BRCA1 expression in yeast.
J. Surg. Res.,113, 62–73.
37. Bergkessel,M. and Reese,J.C. (2004) An essential role for the
Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S
DNA-damage checkpoint recovery. Genetics,167, 21–33.
38. Muhlrad,D. and Parker,R. (1999) Aberrant mRNAs with
extended 30UTRs are substrates for rapid degradation
by mRNA surveillance. RNA,5, 1299–1307.
39. Pedro-Segura,E., Vergara,S.V., Rodriguez-Navarro,S., Parker,R.,
Thiele,D.J. and Puig,S. (2008) The Cth2 ARE-binding protein
recruits the Dhh1 helicase to promote the decay of succinate
dehydrogenase SDH4 mRNA in response to iron deficiency.
J. Biol. Chem.,283, 28527–28535.
40. Decker,C.J., Teixeira,D. and Parker,R. (2007) Edc3p and a
glutamine/asparagine-rich domain of Lsm4p function in
processing body assembly in Saccharomyces cerevisiae.
J. Cell. Biol.,179, 437–449.
41. Goldstrohm,A.C., Hook,B.A., Seay,D.J. and Wickens,M. (2006)
PUF proteins bind Pop2p to regulate messenger RNAs.
Nat. Struct. Mol. Biol.,13, 533–539.
42. Jankowsky,E. and Bowers,H. (2006) Remodeling of
ribonucleoprotein complexes with DExH/D RNA helicases.
Nucleic Acids Res.,34, 4181–4188.
43. Linder,P., Tanner,N.K. and Banroques,J. (2001) From RNA
helicases to RNPases. Trends Biochem. Sci.,26, 339–341.
44. Badis,G., Saveanu,C., Fromont-Racine,M. and Jacquier,A. (2004)
Targeted mRNA degradation by deadenylation-independent
decapping. Mol. Cell.,15, 5–15.
45. Dong,S., Li,C., Zenklusen,D., Singer,R.H., Jacobson,A. and
He,F. (2007) YRA1 autoregulation requires nuclear export and
cytoplasmic Edc3p-mediated degradation of its pre-mRNA.
Mol. Cell.,25, 559–573.
1344 Nucleic Acids Research, 2012, Vol. 40, No. 3
    • By combining different approaches, we distinguished functionally relevant interactions with DDX6 from interactions which depend on the mutual binding of RNA, and protein complex membership. DDX6 is often thought of as a marker of P-bodies [90], and the most well-characterized function of both DDX6 and its yeast ortholog DHH1 is in the mRNA decapping/decay pathway [14,47,88,89,91]. Consistent with this view, we identified a number of P-body/decapping proteins as DDX6 interactors (Table 1).
    [Show abstract] [Hide abstract] ABSTRACT: DDX6 (p54/RCK) is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58) of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2) and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2). We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6's multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6's interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions-many of which are likely conserved across eukaryotes.
    Full-text · Article · Jul 2015
  • [Show abstract] [Hide abstract] ABSTRACT: Translational control is a vital aspect of gene expression. Message specific translational repressors have been known of decades. Recent evidence, however, suggest that a general machinery exists that dampens the translational capacity of the majority of mRNAs. This activity has been best ascribed to a conserved family of RNA helicases called the DHH1 / RCKp54 family. The function of these helicases is to promote translational silencing. By transitioning mRNA into quiescence, DHH1 / RCKp54 helicases promote either mRNA destruction or storage. In this review we describe the known roles of these helicases and propose a mechanistic model to explain their mode of action. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.
    Article · Mar 2013
    Jeff Coller
  • [Show abstract] [Hide abstract] ABSTRACT: Members of the DEAD box family of RNA helicases are known to be involved in most cellular processes that require manipulation of RNA structure and, in many cases, exhibit other functions in addition to their established ATP-dependent RNA helicase activities. They thus play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and/or neoplastic transformation. These proteins generally act as components of multi-protein complexes; therefore their precise role is likely to be influenced by their interacting partners and to be highly context-dependent. This may also provide an explanation for the sometimes conflicting reports suggesting that DEAD box proteins have both pro- and anti-proliferative roles in cancer.
    Article · Apr 2013
  • [Show abstract] [Hide abstract] ABSTRACT: The DEAD box RNA helicase DHH1 acts as a general repressor of translation and activator of decapping but can also act specifically on individual mRNAs. In trypanosomes, DHH1 overexpression or expression of a dhh1 ATPase mutant, dhh1 DEAD:DQAD, resulted in increased or decreased stability of a small group of mRNAs, mainly encoding developmentally regulated genes. Here, four of the mRNAs affected by dhh1 DEAD:DQAD expression have been analyzed to identify cis-elements involved in dhh1 DEAD:DQAD action. For three mRNAs, the 3′ UTR mediated the change in mRNA level and, in one case, both the 5′ and the 3′ UTR contributed. No responsive elements were detected in the protein coding sequences. One mRNA stabilized by dhh1 DEAD:DQAD expression was analyzed in more detail: deletion or mutation of an AU-rich element in the 3′ UTR resulted in mRNA stabilization in the absence of dhh1 DEAD:DQAD and completely abolished the response to dhh1 DEAD:DQAD. While AU-rich instability elements have been previously shown to mediate mRNA decrease or translational exit by recruitment of DHH1, this is, to our knowledge, the first report of an AU-rich instability element that is responsible for a DHH1 mediated increase in mRNA stability. We suggest a novel model for the selective action of dhh1 on individual mRNAs that is based on the change in the turnover rate of stabilizing or destabilizing RNA binding proteins.
    Full-text · Article · Jan 2014
  • [Show abstract] [Hide abstract] ABSTRACT: DDX6 (Rck/p54), a member of the DEAD-box family of helicases, is highly conserved from unicellular eukaryotes to vertebrates. Functions of DDX6 and its orthologs in dynamic ribonucleoproteins contribute to global and transcript-specific messenger RNA (mRNA) storage, translational repression, and decay during development and differentiation in the germline and somatic cells. Its role in pathways that promote mRNA-specific alternative translation initiation has been shown to be linked to cellular homeostasis, deregulated tissue development, and the control of gene expression in RNA viruses. Recently, DDX6 was found to participate in mRNA regulation mediated by miRNA-mediated silencing. DDX6 and its orthologs have versatile functions in mRNA metabolism, which characterize them as important post-transcriptional regulators of gene expression.For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.
    Article · Apr 2014
  • [Show abstract] [Hide abstract] ABSTRACT: Stresses, such as glucose depletion, activate Snf1, the Saccharomyces cerevisiae ortholog of adenosine monophosphate-activated protein kinase (AMPK), enabling adaptive cellular responses. In addition to affecting transcription, Snf1 may also promote mRNA stability in a gene-specific manner. To understand Snf1-mediated signaling, we used quantitative mass spectrometry to identify proteins that were phosphorylated in a Snf1-dependent manner. We identified 210 Snf1-dependent phosphopeptides in 145 proteins. Thirteen of these proteins are involved in mRNA metabolism. Of these, we found that Ccr4 (the major cytoplasmic deadenylase), Dhh1 (an RNA helicase), and Xrn1 (an exoribonuclease) were required for the glucose-induced decay of Snf1-dependent mRNAs that were activated by glucose depletion. Unexpectedly, deletion of XRN1 reduced the accumulation of Snf1-dependent transcripts that were synthesized during glucose depletion. Deletion of SNF1 rescued the synthetic lethality of simultaneous deletion of XRN1 and REG1, which encodes a regulatory subunit of a phosphatase that inhibits Snf1. Mutation of three Snf1-dependent phosphorylation sites in Xrn1 reduced glucose-induced mRNA decay. Thus, Xrn1 is required for Snf1-dependent mRNA homeostasis in response to nutrient availability.
    Article · Jul 2014
Show more

Supplementary resources