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ARTICLES
614 VOLUME 10 NUMBER 8 AUGUST 2003 NATURE STRUCTURAL BIOLOGY
The SAM domain is found in a wide variety of functionally diverse
proteins including regulators of signal transduction and transcription.
SAM domains function as protein-interaction modules through their
well-characterized ability to mediate homo- and heterotypic inter-
action with other SAM domains. For example, SAM domains mediate
the homotypic association of the transcriptional regulators TEL
1
and
polyhomeotic (ph)
2
and the heterotypic association of the MAP kinase
module proteins Ste4 and Byr2 in Schizosaccharomyces pombe
3
or
Ste11 and Ste50 in S. cerevisiae
4
. Indeed, the potent oncogenic proper-
ties of numerous TEL chimeras generated by chromosomal transloca-
tions are attributable in large part to the homo-oligomerization ability
of the TEL SAM domain
5–8
.
To date, the atomic structures of nine different SAM domains have
been reported, representing both monomeric forms
9–12
and higher-
order oligomeric arrangements
13–17
. The 70- to 100-residue SAM
domain consists of four or five α-helices arranged in a globular bun-
dle with a hydrophobic core constituting the most conserved part of
the domain. In contrast, residues on the surface of the domain have
great sequence variability. Higher-order structures of the TEL and ph
SAM domains have revealed a head-to-tail polymer arrangement
16,17
that suggests a likely mechanism for the role of these proteins as tran-
scriptional repressors. Four structures of Eph-receptor tyrosine
kinase SAM domains have been determined representing a
monomeric
11
and three higher-order homotypic arrangements dis-
tinct from the head-to-tail polymer arrangement observed for the
TEL and ph SAM domains
13–15
. The biological relevance of the
higher-order EphB2 and EphA4 SAM domain structures remains to
be determined. As the majority of SAM domains studied until now
seem to be monomeric in solution and potential interacting partners
have not been identified, whether SAM domains function exclusively
to mediate protein-protein interactions has been brought into ques-
tion. Recent studies of the D. melanogaster protein Smaug (Smg)
have suggested a potential role for its SAM domain in RNA recogni-
tion
18,19
.
Smg functions in the proper establishment of the anterior-poste-
rior axis of the developing embryo. The specification of the posterior
pole involves localization of Nanos (Nos) protein to the posterior of
the embryo through the localization of nos mRNA
20–22
. However, the
localization of nos transcripts is an inefficient process and substantial
amounts are found throughout the embryo
23
. Preventing the accu-
mulation of ectopic Nos protein requires the spatial regulation of nos
translation; whereas posteriorly localized nos mRNA is translated,
unlocalized nos mRNA is translationally repressed
24
. Thus, the regu-
lation of translation is coordinated with transcript localization. Smg
represses the translation of unlocalized nos mRNA by interacting with
sequences in the transcript’s 3′ untranslated region (UTR)
18,19,25
.
These RNA sequences have been designated Smg-recognition ele-
ments (SREs). An SRE consists of a stem-loop structure with the loop
sequence CUGGC. Disrupting Smg function leads to the translation
of unlocalized nos mRNA and ectopic Nos protein resulting in lethal
body-patterning defects
19,25
. The RNA-binding activity of Smg maps
to residues 584–763 (ref. 19) and, although sequence analysis of this
region failed to identify a known RNA-binding domain, a SAM
domain was evident between residues 594–655 (ref. 18).
Here we demonstrate that the SAM domain of D. melanogaster Smg
has a direct role in RNA recognition. We have localized the RNA-
1
Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
2
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
3
Department of Biochemistry, University of Toronto,
Toronto, Ontario M5S 1A8, Canada. Correspondence should be addressed to F.S. (sicheri@mshri.on.ca) or C.A.S. (c.smibert@utoronto.ca).
The RNA-binding SAM domain of Smaug defines a new
family of post-transcriptional regulators
Tzvi Aviv
1,2
, Zhen Lin
1
, Stefanie Lau
1
, Laura M. Rendl
3
, Frank Sicheri
1,2
& Craig A Smibert
3
Anteroposterior patterning in Drosophila melanogaster is dependent on the sequence-specific RNA-binding protein Smaug,
which binds to and regulates the translation of nanos (nos) mRNA. Here we demonstrate that the sterile- motif (SAM) domain
of Smaug functions as an RNA-recognition domain. This represents a new function for the SAM domain family, which is well
characterized for mediating protein-protein interactions. Using homology modeling and site-directed mutagenesis, we have
localized the RNA-binding surface of the Smaug SAM domain and have elaborated the RNA consensus sequence required for
binding. Residues that compose the RNA-binding surface are conserved in a subgroup of SAM domain–containing proteins,
suggesting that the function of the domain is conserved from yeast to humans. We show here that the SAM domain of
Saccharomyces cerevisiae Vts1 binds RNA with the same specificity as Smaug and that Vts1 induces transcript degradation
through a mechanism involving the cytoplasmic deadenylase CCR4. Together, these results suggest that Smaug and Vts1 define
a larger class of post-transcriptional regulators that act in part through a common transcript-recognition mechanism.
© 2003 Nature Publishing Group
http://www.nature.com/naturestructuralbiology
ARTICLES
NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 8 AUGUST 2003 615
binding surface of the Smg SAM domain and have elaborated the RNA
consensus sequence required for high-affinity binding. We also
demonstrate that the S. cerevisiae Smg homolog Vts1 binds RNA
through its SAM domain and that Vts1 can regulate the expression of a
target gene in vivo by destabilizing its transcript mRNA. Together this
work defines a new family of SAM domain–containing proteins that
are conserved from yeast to humans and that may function generally
as post-transcriptional regulators in part by binding to common stem-
loop elements in target mRNAs.
RESULTS
A SAM domain–containing fragment of Smg binds RNA directly
Amino acids 583–763 of Smg (Smg(583–763)), containing the SAM
domain, are sufficient for SRE binding
19
. To determine whether the
SAM domain of Smg (Smg
SAM
, residues 594–660) is involved in RNA
binding and to precisely map RNA-binding determinants, we first
generated a homology model using a structure-based sequence align-
ment of SAM domains from different proteins and organisms. A PSI-
BLAST search using Smg
SAM
retrieved numerous SAM
domain–containing proteins as expected. Careful examination of the
resulting sequence alignments (Fig. 1a) revealed a distinct pattern of
conserved, positively charged residues specific to sequences with
E-values <0.42, representing 30–40% sequence identity with Smg
SAM
.
Overall, this identified a cluster of 11 sequences, including hypotheti-
cal human, mouse and Caenorhabditis elegans proteins and fungal
variants of the S. cerevisiae protein Vts1, which represent a subgroup
of SAM domains most closely related to the Smg SAM domain. In
addition to similarities within their SAM domains, two other regions
of similarity are apparent between Smg and 8 of the 11 proteins in this
subgroup. The two regions, designated Smg similarity regions 1 and 2
(SSR1 and SSR2), have been previously noted to be common to Smg
and mouse and human proteins predicted by genomic sequences
18
(Fig. 1b). These observations, supported by the experimental data
provided below, suggest that the group of 11 Smg-like proteins con-
stitute a functionally related family, which we refer to as the Smg
homologs.
Using the EphB2 SAM-domain structure (PDB entry 1F0M, 23%
sequence identity to Smg
SAM
) as a template, a homology model was
constructed for the SAM domain of Smg using SWISS-MODEL
(Figs.1c,d). Notably, residues that are specifically conserved among the
Smg homologs cluster to one surface of the SAM domain. The richness
of basic residues on this surface indicates that it has a potential function
in RNA binding. To test this hypothesis, we assessed the impact of a
number of amino acid substitutions on the ability of Smg(583–763),
our smallest stably expressed fragment containing the SAM domain, to
interact with a fluorescein-labeled model SRE consisting of a stem-loop
structure with a five-nucleotide CUGGC loop (Fig. 2a). Using a fluor-
escence-polarization assay
26
, we found that the model SRE bound to
gi:
dm SMAUG M S G I G L W L
K
SLR L H
K
-YIELF
K
N ---MTYEEMLLITE DFLQ- SVGVT-
K
G
A
S H
K
L
A
LCI D
K
L
K
ER
A
N- ILN 5880909
ag SMAUG M S S I
A
HWL
K
SLR L H
K
- YVWLFSN- - - LTYD
K
ML GI TE EYLQ- SLGVT-
K
G
A
RH
K
L
A
ICIQ
K
L
K
ERYGTLLQ 2129252
6
hs KIAA105
3
STNVP
A
WL
K
SLR L H
K
-Y
A
A
LFSQ---MTYEEMM
A
LTE CQLE-
A
QNVT-
K
G
A
RH
K
IVISIQ
K
L
K
ERQN- L L
K
5689443
mm KIAA105
3
STNVP
A
WL
K
SLR L H
K
-Y
A
A
LFSQ---MTYEEMM
A
LTE CQLE-
A
QNVT-
K
G
A
RH
K
IVISIQ
K
L
K
ERQN- L L
K
26329871
ce SMAUG M R D L G Y W L
K
K
L R L H
K
-Y
A
PLFQD---MTYRQLL SLND NVLE- RM
K
VT- NG
A
R
K
K
I
A
QSI E
K
LYERP
A
- LLR 2515001
6
hs BDG-29 Q N G I L D W L R
K
L R L H
K
- YYPVF
K
Q ---LSME
K
FLSLTE EDLN-
K
FESLTMG
A
K
K
K
L
K
TQLELE
K
E
K
SE- RRC 18146833
mm BDG-29 Q N G I L D W L R
K
L R L H
K
- YYPVF
K
Q ---LTME
K
FLSLTE EDLN-
K
FESLTMG
A
K
K
K
L
K
TQLELE
K
E
K
SE- RRC 18252798
ca VTS1 L N N I P
A
WL
K
LLR L H
K
-YTECL
K
D ---VPW
K
ELIELDNDQLE- S
K
GV
A
A
LG
A
RR
K
LL
K
A
FDVV
K
NNLP-VV 3859708
sp VTS1 P Q DI PSWLR SLR L H
K
-YTNNL
K
D ---TDWD
A
LVSLSD LDLQ- NRGI M
A
LG
A
RR
K
LL
K
SFQEV
A
PLVS- S
K
K
7688323
sc VTS1 L K N I P M W L K SLR L HK-YSDALSG---TPWIELIYLDD ETLE- KKGVLALGARRKLLKAFGI VI D YKE- RDL 1420780
hs EPHB2 F N T V D E W L E
A
I
K
MGQ- Y
K
ESF
A
N
A
G FTSFDVVSQMMMEDI L- RVGVTL
A
GHQ
K
K
ILNSIQVMR
A
QMNQIQ 9256876
hs EPHA
4
VVSVGDWLQ
A
I
K
MDR -Y
K
DNFT
A
A
GYTT L E
A
VVHMSQDDL
A
-RIGIT
A
ITHQN
K
ILSSVQ
A
M R TQMQQMH 492986
4
sp BYR2 S
K
EV
A
EWL
K
SI GLE
K
-YIEQFSQNNIEG-RHLNHLTLPLL
K
-DLGIENT
A
K
G
K
QFL
K
QRDYLR EFPRPCI 101060
dd KYK1 P N D V
A
IWLESFNYGQ- YR
K
NFRDNNI SG- RHLEGI TH
A
ML
K
NDLGI EPYGHR EDI I NRLNRMI QI WND
K
S 1730077
sp STE4 N E
A
VCNWI EQLGFP- - H
K
E
A
FEDYHI LG-
K
DI DLLSSNDLR- DMGI ESVGHR IDILS
A
IQSM
K
K
QQ
K
D
K
L 548999
sc BOB1 P E E V T D Y F S L V G F D Q S T C N
K
F
K
EHQVSG-
K
ILLELELEHL
K
-ELEINSFGIR FQI F
K
EI RNI
K
S
A
IDSSS 584850
dm PH V D D VSNFI RELPGCQDYVDDFI QQEI DG- Q
A
LLLL
K
E
K
HLVN
A
MGM
K
-LGP
A
L
K
IV
A
K
VESI
K
EVPPPGE 730323
sc STE11 L P F V Q L F L E E I G C T Q - Y L D S F I Q C N L V T E E E I
K
YLD
K
DI LI -
A
LGVN
K
IGDRL
K
ILR
K
S
K
SFQRD
K
RI EQ 1711551
**
600
pdb id: 1F0M
*
*
**
610
620
630 640 650
660
Smg SAM
α1 α2 α3 α5
α4
SAM
SAM
SAM
SAM
SAM
S
S
R1
S
S
R1
S
SR1
S
S
R
2
S
S
R2
Zif
hsBDG-29
mmBDG-29
ceSMAUG
hsFLJ10122
mmKIAA1053
hsKIAA1053
dmSMAUG
agSMAUG
spVTS1
scVTS1
caVTS1
N
C
Tyr
613
Lys
645
Lys
612
Arg
609
Lys
606
His
611
Ala
642
α1
α1
α2
α2
α3
α3
α4
α4
α5
α5
Figure 1 Homology modeling of Smg SAM domain. (a) Sequence alignment of SAM domains of Smg homologs and selected proteins. GenBank accession
numbers are indicated on the right. Secondary structure elements corresponding to the EphB2 SAM domain crystal structure are indicated. Conserved
hydrophobic residues, green; acidic residues, red; basic residues, blue. Mutations in Smg or Vts1 that perturbed SRE binding, red stars; benign mutation,
green diamond. Species abbreviations: dm, D. melanogaster; ag, Anopheles gambiae; hs, Homo sapiens; mm, Mus musculus; ce, C. elegans; ca, Candida
albicans; sp, S. pombe; sc, S. cerevisiae; dd, Dictyostelium discoideum. (b)Cladogram representing overall sequence similarity and domain architecture
of the Smg homologs. See text for description of SAM, SSR1 and SSR2 domains. Zif, CCHC zinc-finger domain (SMART
43
). (c,d) Ribbon and surface
representations of the Smg SAM domain, respectively. In c, secondary structure elements encompassing RNA-binding surface are pink and conserved side
chains specific to Smg homologs are in ball-and-stick representation. In d, regions corresponding to conserved basic and hydrophobic residues specific to
the Smg homologs are blue and green, respectively.
a
b
c
d
© 2003 Nature Publishing Group
http://www.nature.com/naturestructuralbiology
ARTICLES
616 VOLUME 10 NUMBER 8 AUGUST 2003 NATURE STRUCTURAL BIOLOGY
Smg(583–763) with high affinity (K
d
= 40 nM) whereas mutants within
the Smg conserved surface were greatly deficient for binding.
Specifically, the mutations K612Q, A642H, A642Q and the double
mutation K606A R609A completely abolished RNA binding (Fig. 2b,
Ta b le 1) whereas the single point mutations R609A, K606A, Y613F and
H611Q reduced binding three- to ten-fold. None of these mutations
perturbed the overall fold of the proteins as assessed by circular-
dichroism spectroscopy (see Supplementary Fig. 1 online) and elution
volume during size-exclusion chromatography (data not shown).
These results indicate that the SAM domain of Smg does have a role in
RNA binding and localizes a putative RNA-binding surface to a region
encompassing the C terminus of helix α1, the connecting segment
between helix α1 and α2 and the N terminus of helix α5 (Fig. 1c,d).
Notably, this delineated surface is distinct from the homo-oligomeriza-
tion surfaces identified for the Eph-A4- and Eph-B2-receptor SAM
domains and partly overlaps with one of the two surfaces (the EH sur-
face) that mediate head-to-tail polymer formation by the TEL and ph
SAM domains.
The presence of an additional ∼100 amino acids C-terminal to the
SAM domain in Smg(583–763) suggested that the SAM domain may
not bind RNA directly and that the mutations characterized as dis-
rupting RNA binding may act in an indirect manner (for example by
disrupting a protein-oligomerization surface that is indirectly required
for RNA-binding function). A detailed functional characterization of
the yeast Smg homolog Vts1 described below resolved this ambiguity.
Vts1 is an RNA binding protein
The finding that mutation of residues in Smg that are conserved
among the Smg homologs abolished RNA binding strongly suggested
that other Smg homologs would also bind to stem-loop RNAs. To test
this hypothesis, we focused our efforts on the S. cerevisiae protein
Vts1. VTS1 is a nonessential gene whose protein product has been
implicated in vesicular transport on the basis of its ability to suppress
the growth and vacuolar-transport defects in vti1-2 cells carrying a
temperature-sensitive allele of Q-SNARE VTI1
27
. To test the ability of
Vts1 to bind RNA, we overexpressed and purified a FLAG-tagged
version of full-length Vts1 from S. cerevisiae. This protein binds
our model SRE with high affinity (K
d
= 17 nM) as assessed by a
fluorescence-polarization assay (Fig. 2c, Tabl e 1). To assess whether
the same SAM-domain surface of Vts1 is important for RNA binding,
we introduced the mutations K467Q and A498Q (analogous to Smg
mutations K612Q and A642Q) into the VTS1 FLAG-tagged expression
construct. Both mutations perturbed Vts1 binding to the model SRE,
although the K467Q mutation did not have as marked an effect as did
the analogous mutation in Smg (Figure 2c, Tabl e 1). Together, these
results demonstrate that Vts1 also binds to RNA stem-loop structures
in a SAM domain–dependent manner.
The SAM domain of Vts1 binds RNA directly
We have expressed a 13-kDa protein fragment of Vts1 consisting
solely of its SAM domain (Vts1
SAM
, residues 407–523). As assessed by
our in vitro fluorescence-polarization assay (Fig. 2d, Tab le 1), this
protein fragment is sufficient for high-affinity binding to our model
SRE (K
d
= 14 nM). In addition, RNA binding by Vts1
SAM
is disrupted
by the same SAM-domain mutations, K467Q and A498Q, that per-
turb RNA-binding function of full-length Vts1 and Smg(583–763).
These results demonstrate that the Vts1 interaction with RNA is
mediated directly by its SAM domain. As Smg(583–763) and Vts1
SAM
share similarity only within their SAM domains (Vts1
SAM
lacks the
100-amino-acid C-terminal extension of Smg(583–763)), these
results also suggest that the RNA-binding function of Smg is medi-
ated directly by its SAM domain.
Additional studies using size-exclusion chromatography and static
light-scattering analysis revealed that Vts1
SAM
is a monomer in solu-
tion in the absence of RNA and that in the presence of molar excess of
either of two different model SRE RNAs, the domain forms 1:1 stoi-
chiometric complexes (Tab le 2). These observations rule out a
U
C
G
G
A
*
*
*
*
A
G
U
C
U
C
U
G
G
C
1
2
3
4
5
6
7
8
9
1
2
3
4
5
wt
R609A
A642Q
K612Q
K606A/
R609A
A642H
K606A
Y613F
H611Q
10
–1
10
0
10
1
10
2
10
3
10
4
10
5
100
150
200
250
[Smg(583–763)] (nM)
Polarization (mP)
10
(–)
(–)
(–)
(–)
(–)
wt
A498Q
K467Q
10
0
10
1
10
2
10
3
100
150
200
250
Polarization (mP)
10
–1
10
0
10
1
10
2
10
3
10
4
10
5
100
150
200
Polarization (mP)
[Vts1(1–523)] (nM)
[Vts1
SAM
] (nM)
Figure 2 Protein determinants of the Smg and Vts1 SAM domains required
for SRE binding. (a) Schematic of fluorescein-labeled model SRE, based on
the nos 3′ UTR
27–41
. Bases are numbered from first position of loop;
asterisks indicate expected Watson-Crick base pairing in the stem. Bases
required for Smg binding, bold. (b) Mutational and SRE binding analysis of
Smg(584–763). (c) Mutational and SRE binding analysis of full-length
Vts1(1–523). (d) Mutational and SRE binding analysis of Vts1
SAM
.
Table 1 SRE
a
binding to Smg and Vts1 mutants
Protein K
d
(nM) 95% confidence
interval
Smg(583–763)
WT 40 33–48
K606A 380 290–497
R609A 225 209–243
H611Q 127 114–142
K612Q NB –
Y613F 337 300–378
A642H NB –
A642Q NB –
K606A R609A NB –
Vts1(1–523)
WT 17 14–22
K467Q 91 71–117
A498Q NB –
Vts1(407–523)
WT 14 8–24
K467Q NB –
A498Q NB –
a
SRE, fluorescein-5′-AGGCUCUGGCAGCCU3′. WT, wild type. NB, no binding.
a
b
c
d
© 2003 Nature Publishing Group
http://www.nature.com/naturestructuralbiology
ARTICLES
NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 8 AUGUST 2003 617
requirement for SAM domain–mediated protein-protein interactions
in the RNA-binding event and demonstrate that the functional surface
delineated by mutational analysis of Smg and Vts1 comprises a bona
fide RNA-binding surface.
The Smg RNA-binding consensus
The observation that similar SAM-domain mutations disrupted the
binding of Vts1 and Smg to our model SRE suggested that the two
proteins interact with RNA through a common mechanism. To con-
firm this possibility, we examined the RNA-binding specificity of
both Smg and Vts1. We first performed an extensive mutational
analysis on our model SRE to determine the consensus sequence for
Smg binding. Work until now had suggested that the nucleotides
required for sequence-specific binding reside within the loop
19,25,28
.
Thus, we comprehensively replaced each of the five loop bases in our
model SRE (Fig. 2a) with the three other possible bases and tested
each for binding to Smg(583–763) using a fluorescence-polarization
assay. We found that changes to the second or the fifth base of the SRE
loop had little to no effect on Smg binding (Fig. 3a, panel 1; Ta ble 3).
In contrast, all mutations to the first, third and fourth bases of the
loop either abolished binding or reduced it substantially (Fig. 3a,
panels 2 and 3; Ta ble 3). Using a gel-shift assay, we observed a similar
trend of binding for all mutant SREs tested (Supplementary Fig. 2
online). Thus, these analyses reveal a strict binding requirement for
positions 1, 3 and 4 but not for positions 2 and 5 of the model SRE. To
further examine the functional role of loop positions 2 and 5, the
bases at these positions were deleted to make four-base loops. The
deletion of position 2 from the SRE loop abolished Smg binding
whereas the deletion of position 5 did not (Fig. 3a, panel 4; Ta ble 3).
These results demonstrate that there is a structural requirement for
separation between loop positions 1 and 3, whereas no such separa-
tion is required between loop position 4 and the stem. We also
assessed the requirement of the SRE stem for Smg binding. The two
stem mutants (–1)U/6U and (–2)C/7C, which were predicted to dis-
rupt Watson-Crick base-pairing, abolished binding (Fig. 3a, panel 4;
Ta ble 3;see Fig. 2a for model SRE numbering scheme). Smg binding
to the stem mutants could be rescued by compensatory mutations
that restored base pairing in the stem ((–1)A/6U and (–2)G/7C ,
Fig. 3a, panel 4; Ta ble 3). These results confirm the previously
observed requirement of a nonspecific stem for Smg binding
25,28
.
Ta ken together, our mutagenesis data define an RNA sequence con-
sensus for Smg binding consisting of a stem-loop structure with either
a four- or five-base loop. The loop consists of the sequence CNGG or
CNGGN, where N is any base. Currently the nos transcript is the only
known target of Smg, but the phenotype of embryos derived from smg
mutant mothers suggests that Smg may regulate the expression of
additional mRNAs
19
. The SRE consensus that we have delineated may
help in the identification of other Smg targets.
Vts1 interacts with RNA through a similar mechanism
After defining the RNA consensus for Smg, we assessed the RNA-bind-
ing specificity of Vts1. As observed for Smg(583–763), we found that
full-length Vts1 did not bind to model SREs mutated at positions 1, 3,
and 4 but did bind to SREs mutated at positions 2 and 5 (Fig. 3b,
Ta b le 3 ). These data suggest that Vts1 and Smg have similar if not
identical RNA-binding specificities. The finding of identical RNA and
protein determinants required for high-affinity interaction suggests
that Smg and Vts1 interact with stem-loop RNAs through a common
mechanism. As VTS1 is the most divergent of the 11 Smg homologs
(Fig. 1b), these results also strongly suggest that all 11 Smg homologs
identified in this study will bind to similar stem-loop RNA structures
through a common mechanism.
VTS1 regulates expression of target mRNAs in vivo
Given that Vts1 is an RNA-binding protein, we next set out to deter-
mine whether VTS1 could regulate the expression of a reporter gene
that bears SREs in vivo. To do so, we used a galactose-inducible green
(1) (2)
(3)
wt
2A
2G
2C
5A
5U
5G
10
–1
10
0
10
1
10
2
10
3
10
4
10
5
100
150
200
250
Polarization (mP)
wt
4U
3A
3U
1G
1U
10
–1
10
0
10
1
10
2
10
3
10
4
10
5
100
150
200
250
wt
4C
4A
3C
1A
10
–1
10
0
10
1
10
2
10
3
10
4
10
5
100
150
200
250
Polarization (mP)
[Smg(583–763)] (nM)
(–)1U/6U
(–)1A/6U
wt
(–)2C/7C
∆
5
∆
2
(–)2G/7C
(4)
10
–1
10
0
10
1
10
2
10
3
10
4
10
100
150
200
250
5
3
wt
5U
2A
4A
3C
1G
4U
∆5
10
0
10
1
10
2
10
100
150
200
250
Polarization (mP)
[Vts1(1–523)] (nM)
[Smg(583–763)] (nM)
Figure 3 RNA stem-loop determinants for binding to Smg and Vts1.
(a) Binding analysis of wild-type and mutant SREs to Smg(584–763). (1)
SRE mutants in loop positions 2 and 5. (2,3) SRE mutants in loop positions
1, 3 and 5. (4) SRE loop deletions in positions 2 and 5 and stem mutants.
(b) Binding analysis of wild-type and mutant SREs to Vts1(1–523).
Table 2 Static light scattering analysis of Vts1 and Vts1–RNA
complexes
Theoretical SLS
monomer observed Stoichiometry
mass mass
Lysozyme standard 14,300 15,660 ± 3% Monomer
RNA standard
a
4,783 9,217 ± 8% Dimer
Vts1
SAM
12,935 12,860 ± 5% Monomer
Vts1
SAM
12,935 13,320 ± 5% Monomer
RNA_6
b
5,059 5,056 ± 13% Monomer
RNA_7
c
5,067 6,116 ± 15% Monomer
Vts1
SAM
+ RNA_6
b
17,994 17,210 ± 5% 1:1
Vts1
SAM
+ RNA_7
c
18,002 17,779 ± 5% 1:1
Unit for molecular mass in Da; SLS, static light scattering.
a
RNA standard, 5′-UGGGCUCUGGAGCCC-3′.
b
RNA_6, 5′-CAUCUCUGGCAGAUGU-3′.
c
RNA_7, 5′-AUAUCUCUGGCAGAUA-3′.
a
b
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ARTICLES
618 VOLUME 10 NUMBER 8 AUGUST 2003 NATURE STRUCTURAL BIOLOGY
fluorescent protein (GFP) reporter gene that expresses a transcript
bearing three SREs in its 3′ UTR (3xSRE
+
; Fig. 4a). The 3xSRE
+
ele-
ment mediates Smg-dependent regulation of protein expression both
in vivo and in vitro
18,25
. As a control, a similar GFP reporter gene was
constructed (GFP-3xSRE
–
) that expresses transcripts in which each
SRE was mutated in a manner that eliminates Smg binding, thus
blocking Smg-dependent regulation.
After transformation of S. cerevisiae with the GFP-3xSRE
+
and
GFP-3xSRE
−
reporter constructs, cells were analyzed by fluorescence
microscopy. We found that the number of cells expressing detectable
levels of GFP was substantially higher when the cells carried the
GFP-3xSRE
–
plasmid as compared with the GFP-3xSRE
+
plasmid
(data not shown). This suggested that the presence of functional
SREs in the 3′ UTR of the transcript inhibits GFP expression. To
quantify this effect, we used flow cytometry to measure levels of GFP
fluorescence. We found that although the levels of GFP fluorescence
varied over a broad range, the overall level of fluorescence associated
with GFP-3xSRE
+
cells was substantially lower than that associated
with GFP-3xSRE
–
cells (Fig. 4a). To determine if this repression was
VTS1 dependent, both reporter genes were introduced into a vts1
deletion strain (vts1∆). In vts1∆ cells, both the GFP-3xSRE
+
and
GFP-3xSRE
–
reporter genes were expressed at similar levels,
comparable to that of the GFP-3xSRE
–
reporter gene in wild-type
cells (Fig. 4a). Taken together, these results suggest that the Vts1
protein interacts with SRE-bearing mRNAs in vivo and acts to
repress reporter-gene expression.
Smg represses gene expression by inhibit-
ing translation
18,19,25
. To determine how
Vts1 represses gene expression, we first com-
pared the steady-state levels of the reporter
gene mRNAs by northern blot analysis. We
found that GFP-3xSRE
–
mRNA was about
two-fold more abundant than the GFP-
3xSRE
+
mRNA in wild-type cells, but that
both transcripts were equally abundant in
vts1∆cells (Fig. 4b). This suggested that Vts1
acts to destabilize the GFP-3xSRE
+
tran-
script. To confirm this possibility, we mea-
sured the half-lives of the GFP-3xSRE
+
and
GFP-3xSRE
–
reporter gene transcripts. In
this experiment, transcription of the
reporter gene was briefly induced with galac-
tose and then inhibited by the addition of
glucose. Reporter-gene expression was then
measured at short time intervals by northern
blotting. The GFP-3xSRE
+
mRNA levels
were substantially reduced immediately after
addition of glucose (Fig. 4c). In contrast,
GFP-3xSRE
–
mRNA persisted for a consider-
ably longer period of time (Fig. 4c). Again,
this effect was VTS1 dependent as the stabil-
ity of the GFP-3xSRE
+
mRNA was enhanced
in a vts1∆ strain relative to the GFP-3xSRE
–
mRNA (Fig. 4d). These results clearly
demonstrate that Vts1 induces the degrada-
tion of target mRNAs.
To investigate the mechanism by which
VTS1 influences mRNA stability, we ana-
lyzed the expression of our GFP-3xSRE
+
and
GFP-3xSRE
–
reporter constructs in several
yeast strains that carry deletions in known
mRNA decay factors
29
including kem1 (xrn1), a 5′→3′ exonuclease,
ski2 and ski8, which are involved in 3′→5′ exonucleolytic decay, and
ccr4, pop2 and pan3,which are involved in mRNA de-adenylation.
Yeast contain two deadenylase activities each consisting of multipro-
tein complexes. Ccr4 is the catalytic subunit of one of these com-
plexes, which also includes Pop2 (refs. 30,31). The other
deadenylase, poly(A) nuclease (PAN), consists of the Pan2 and Pan3
proteins
32
. SRE-dependent repression of reporter-gene expression is
abolished in ccr4∆ cells and partially abated in pop2∆ cells, whereas
repression is maintained in all other deletion strains tested (Fig. 5a).
The involvement of CCR4 in SRE-mediated repression of reporter
gene expression is further supported by steady-state analysis of GFP-
3xSRE
+
and GFP-3xSRE
–
transcript levels (Fig. 5b). In contrast to
wild-type cells, which exhibit 0.54 ± 0.06-fold lower levels of GFP-
3xSRE
+
versus GFP-3xSRE
–
transcript levels, ccr4∆ cells exhibit sim-
ilar transcript levels (1.1 ± 0.2-fold difference) comparable to those
observed for vts1∆ cells (0.93 ± 0.1-fold difference). In pop2∆ cells, a
partial attenuation of repression for GFP-3xSRE
+
versus GFP-
3xSRE
–
transcripts levels is also observed (0.7 ±0.03-fold difference).
Taken together these results suggest that VTS1 may influence tran-
script mRNA stability by regulating poly(A) tail length in a CCR4-
dependent manner. Interestingly, the CCR4 deadenylase is also
involved in Puf3-mediated degradation of mRNAs. Puf3 is a
sequence-specific RNA-binding protein that destabilizes COX17
mRNA by recognizing cis-acting elements within the COX17 tran-
script’s 3′ UTR
31
. Thus, two unrelated RNA-binding proteins seem
G
F
P
SC
R
1
SC
R
1
1
SC
R
1
1
0
2 4
6
8
12 1
8
024 681218
0
2
4
6
8
12
18
Wild-t
y
p
e
vts1∆
Steady state
<1
~
6
~4
~4
Time
(
min
):
t
1/2
t
1/2
GFP fluorescence
Cell counts
WT vts1∆
GFP
5'
3'
3'
GFP-3xSRE
+
GFP-3xSRE
−
= CUGGC loop
= GUCGC loop
10
0
10
1
10
2
10
3
10
4
0
20
40
60
80
100
GFP-3xSRE
+
:WT/GLU
GFP-3xSRE
−
:WT/GLU
GFP-3xSRE
+
:WT
GFP-3xSRE
+
:vts1∆
GFP-3xSRE
−
:WT
GFP-3xSRE
−
:vts1∆
GFP
5'
GFP-3xSRE
G
−
GFP
-
3xSRE
G
+
GFP-3xSRE
G
−
GFP-3xSRE
G
+
GFP-3xSRE
−
−
GFP-3xSRE
+
+
SCR1
SCR1
Time (min):
GFP-3xSRE
−
GFP-3xSRE
+
Figure 4 VTS1 regulates gene expression by destabilizing SRE-bearing transcripts. (a) Schematic
representation of GFP reporter constructs (top) and flow cytometry analysis (bottom) of 10,000 wild-
type or vts1∆ yeast cells expressing either GFP-3xSRE
+
or GFP-3xSRE
–
reporter constructs. GFP-
3xSRE
+
GLU and GFP-3xSRE
–
GLU profiles represent 2% (w/v) glucose control cultures (see Methods).
(b) Northern blot analysis of steady-state levels of GFP-3xSRE
+
and GFP-3xSRE
–
mRNAs in wild-type
and vts1∆ yeast strains. (c,d) Northern blot time-course analysis of GFP-3xSRE
+
and GFP-3xSRE
–
mRNAs in wild-type (c) and vts1∆ (d) yeast strains. Approximate half-lives are indicated in minutes.
ab
c
d
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NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 8 AUGUST 2003 619
to use the same molecular mechanisms that induce the degradation
of target mRNAs.
DISCUSSION
Herein, we provide evidence that the SAM domains of Smg and Vts1
bind RNA through a common mechanism. This finding identifies an
altogether new function for a SAM domain. As with the head-to-tail
polymerization mechanism shared by the SAM domains of TEL and
ph transcription factors, the RNA-binding mechanism that we have
identified for Smg and Vts1 seems specific to a limited subgroup of
SAM domain–containing proteins. Indeed, we could not detect SRE-
binding activity for the SAM domains of S. cerevisiae Ste11 and Ste50
or for the human EphA4-receptor tyrosine kinase (data not shown).
Furthermore, the conservation of SAM-domain residues required by
Smg and Vts1 for high-affinity RNA binding strongly suggests that
SRE recognition is an activity specific to at least the 11 Smg homologs
identified in this study. As the vast majority of SAM domains in char-
acterized proteins lack the key residues required for SRE recognition
and have no ascribed protein-protein interaction function, this may
indicate that other new SAM-domain functions remain to be discov-
ered. Notably, our observation that the SAM domain can also function
as an RNA-binding module is reminiscent of the behavior of another
architecturally distinct protein domain. As first structurally character-
ized in karyopherin-α
33
and in β-catenin
34
, the Armadillo repeat
domain was shown to function as a protein-interaction module
through its ability to recognize linear peptide epitopes. Subsequently,
the same domain architecture (also called the Puf domain) was shown
to function using the same molecular interaction surface as that of an
RNA-binding domain in Pumilio, which, like Smg, functions as a
translational repressor in the early D. melanogaster embryo
35,36
.
Our results suggest that Smg and Vts1 define a broader functional
class of SAM domain–containing proteins that may regulate gene
expression post-transcriptionally in part through their common abil-
ity to bind stem-loop structures in target mRNAs. Once bound to their
respective targets, Smg and Vts1 probably rely on protein-protein
interactions involving regions outside of the SAM domain to influence
mRNA stability and translation. Consistent with the importance of
regions outside of the SAM domain are experiments showing that a
transgene expressing the Smg SAM domain plus a short C-terminal
extension fails to rescue the smg mutant phenotype
19
.
On a cursory level, the underlying mechanisms used by Smg and
Vts1 to regulate gene expression once bound to mRNA seem com-
pletely distinct (translational repression versus mRNA stability,
10
0
10
1
10
2
10
3
10
4
ski2
∆
2∆
ccr4
∆
4∆
0
20
40
60
Wild t
y
p
e
v
t
s1
∆
∆
pop2
∆
2∆
10
0
10
1
10
2
10
3
10
4
pan3
∆
3∆
0
20
40
60
0
20
40
60
Cell counts
k
e
m
1
∆
10
0
10
1
10
2
10
3
10
4
ski8
∆
8∆
GFP fluorescence
0.0
0.5 1.0 1.5
vts1∆
ccr4∆
pop2∆
kem1∆
pan3∆
ski2∆
ski8∆
GFP-3xSRE
+
/GFP-3xSRE
−
Genetic background
wt
Figure 5 VTS1 regulation of gene expression is CCR4 dependent. (a) Flow
cytometry analysis of GFP protein levels expressed from GFP-3xSRE
+
(solid
profile) and GFP-3xSRE
–
(line profile) reporters in yeast strains deficient for
mRNA decay factors. (b) Corresponding ratios of steady-state mRNA levels
of GFP-3xSRE
+
and GFP-3xSRE
–
reporters in yeast strains deficient for
mRNA decay factors. Ratios are derived from northern blot analysis (as in
Fig. 4b).
Table 3 Smg and Vts1 binding to SRE mutants
Protein SRE
a
K
d
(nM) 95% confidence
interval
Smg(583–763) WT 13 8–23
2A 15 8–29
2G 80 41–160
2C 23 11–46
5A 48 29–81
5U 31 23–41
5G 24 10–56
∆57441–132
(–1)A/6U 36 20–67
(–2)G/7C 27 15–48
1G 1,600 670–3,900
1U 5,000 580–43,000
3A 1,700 580–5,000
3U 3,300 1,400–8,300
4U 5,400 920–32,000
(–1)U/6U 1,545 700–3,418
1A NB –
3C NB –
4C NB –
4A NB –
∆2NB–
(–2)C/7C NB –
Vts1(1–523) WT 18 14–22
2A 16 11–23
5U 10 5–20
∆52118–23
1G NB –
3C NB –
4A NB –
4U NB –
a
SRE, fluorescein-5′-AGGCUCUGGCAGUCU3’. WT, wild type. NB, no binding.
a
b
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ARTICLES
620 VOLUME 10 NUMBER 8 AUGUST 2003 NATURE STRUCTURAL BIOLOGY
respectively). However, given that Vts1 seems to function through a
poly(A) tail–related mechanism and that the poly(A) tail has a central
role in both translation and mRNA stability
37
, it remains possible that
Smg and Vts1 use a related mechanism to regulate the expression of
target mRNAs. Notably, reporter transcripts bearing functional SREs
are destabilized in early D. melanogaster embryos
25
and nos mRNA is
stabilized in embryos derived from smg mutant mothers in an SRE-
dependent fashion (J.L. Semotok, H.D. Lipshitz and C.A.S., unpub-
lished data). Although these results might suggest that Smg does
indeed regulate mRNA stability, this effect could be a secondary con-
sequence of Smg’s role in translation regulation. An additional com-
plication is that expression of nos mRNA does not seem to be
regulated by changes in poly(A) tail length
38
. Hence, further study of
the underlying post-transcriptional regulatory mechanisms is
required to discern the full extent of functional conservation across
the Smg homolog family.
Although the only known target of Smg in D. melanogaster is nos,
the SRE of nos is not conserved in the mammalian nos homologs, and
in addition, a nos homolog is not evident in fungal genomes. Hence
the actual targets of the human and fungal Smg homologs are
unknown. On the basis of statistical probabilities, the RNA consensus
required for Smg and Vts1 binding is expected to occur once in every
10,000–100,000 bases. This may indicate that the Smg homologs regu-
late a large number of mRNAs; indeed, ∼18% of full-length
D. melanogaster cDNA sequences (1,155 out of 6,147 examined)
contain at least one putative SRE with a minimal five-base-pair stem.
Alternatively, the RNA consensus sequence that we have delineated
may not be sufficient to confer target specificity in vivo and other RNA
binding activities may be at play. For example, 2 of the 11 Smg
homologs identified in this study have a putative zinc-finger RNA-
binding domain, which may act in this regard (Fig. 1b). Alternatively,
the presence of multiple SREs within a transcript RNA may have a role
in defining target selection and indeed, the Smg target nos bears two
SREs in its 3′ UTR. However, the function of this redundancy in nos
mRNA has yet to be explored. A requirement for multiple SREs may
result from the fact that in addition to the SAM domain, an SSR1
domain is found in 8 of the 11 Smg homologs. This domain functions
as a dimerization domain in the context of the F-box adapter proteins
βTRCP
39
, Pop1-Pop2 (ref. 40) and Cdc4 (F.S., unpublished data).
Ultimately, the fully elaborated set of rules that define RNA recogni-
tion by the Smg homologs in vivo will be revealed with the identifica-
tion of the biological targets of these proteins.
METHODS
Sequence alignment and homology modeling. Smg homologs were identified
in a nonredundant database (NCBI) using PSI-BLAST with the Smg SAM
domain (residues 594–660). The sequence alignment of full-length Smg
homologs (E-value < 0.42) was done with ClustalX
41
and the cladogram
(Fig. 1b) was generated using TreeView
42
. For homology modeling, SAM
domains of the Smg homologs and representative SAM domains from the
SMART database
43
were aligned using ClustalX and then refined manually.
The resulting alignment and the EphB2 SAM-domain crystal structure (PDB
entry 1F0M) were then employed using SWISS-MODEL
44
to generate a
refined coordinate model for Smg
SAM
. Ribbon and surface representations
were generated using RIBBONS
45
and GRASP
46
, respectively.
Protein expression, purification and mutagenesis. Smg(583–763) harboring a
stabilizing mutation (C649A) and Vts1
SAM
were expressed as GST fusions in
E. coli using the pGEX-2T expression vector (Pharmacia). Bacterial pellets were
lysed in a solution containing 10% (v/v) glycerol, 750 mM NaCl, 20 mM
HEPES, pH 7.5, 1% (w/v) Sarkosyl, 4 mM DTT, 2 mM phenylmethylsulfonyl-
fluoride and 1.5 µM ethidium bromide. Proteins were purified using
gluthathione-Sepharose affinity chromatography and eluted by thrombin
cleavage. Eluted protein was concentrated and loaded on a Superdex-75 size
exclusion column (Pharmacia) for final purification and characterization (siz-
ing column buffers: 300 mM NaCl, 20 mM HEPES, pH 7.5 for Smg(583–763)
and 100 mM NaCl, 20 mM HEPES, pH 7.5 for Vts1
SAM
). Proteins were concen-
trated to ∼20 mg ml
–1
(as determined by A
280
measurements and theoretical
extinction coefficients); aliquots were flash frozen in liquid nitrogen and then
stored at –80 °C. FLAG-tagged S. cerevisiae Vts1(1–523) was cloned and puri-
fied as described for the genome-wide proteomics analysis
47
. The QuikChange
kit (Stratagene) was used to generate all site-directed mutants.
Fluorescence-polarization assay. 5′-fluorescein-labeled SRE and SRE mutants
were synthesized and then deprotected and desalted as recommended by the
manufacturer (Dharmacon). Before binding analysis, RNAs were heated to
95 °C for 5 min and immediately cooled on ice for 5 min. The fluorescence-
polarization assay used in this study to quantify protein-RNA affinities was
essentially the same as that described for quantifying protein-DNA affinities
26
.
Purified proteins were serially diluted in binding buffer containing 20% (v/v)
glycerol, 150 mM NaCl, 20 mM HEPES, pH 7.0, and ∼2,000 intensity units of
fluorescein-labeled RNA (∼1 nM). Binding reactions were incubated at room
temperature for 10 min. Anisotropy measurements were taken at 22 °C on
100-µl samples using a Beacon 2000 fluorescence polarization instrument. All
measurements were taken in duplicate. Dissociation constants were estimated
by nonlinear regression to a single-site binding model using GraphPad Prism
(GraphPad Software).
Static light-scattering analysis. Static light-scattering experiments were done at
room temperature using a liquid chromatography system consisting of a
Shimadzu LC 10AD-VP isocratic pump, Rheodyne injector, Pharmacia
Superdex 75 HR 10/30 column (24-ml bed volume), Wyatt MINI DAWN static
light scattering detector and GBC LC1240 refractive index detector. Aqueous
buffer system (0.02-µm filtered) consisted of 20 mM HEPES, 150 mM KCl,
1 µM NaN
3
. Refractive index and light scattering detectors were calibrated
using lysozyme (14.3 kDa) and a ‘non-SAM domain–binding’ RNA oligomer
that forms a tight dimer in solution. Protein samples (100 µl) alone or with
two-fold molar excess of model RNAs were loaded at ∼10 mg ml
–1
and chro-
matograms were developed at 0.3 ml min
–1
. Data acquisition and analysis was
carried out using ASTRA version 4.0 as described by the manufacturer (Wyatt
Te c hnology). Smg(583–763) was refractory to static light-scattering analysis
because of its poor solubility characteristics at room temperature.
Flow cytometry and northern blot analysis. A parental reporter gene was con-
structed by insertion of a GFP coding sequence downstream of GAL1 pro-
moter in a centromeric vector bearing a HIS3 selection marker (p413-GFP).
To generate GFP-3xSRE
+
and GFP-3xSRE
–
derivative constructs, a 3xSRE
+
or
3xSRE
–
fragment
25
was inserted into the parental GFP reporter 3′ UTR. For
reporter analysis, yeast strains (see Supplementary Table 1 online for strain
descriptions) were transformed and grown to mid-log phase in 10 ml SC –His
medium containing 2% (w/v) raffinose. GFP expression was induced by the
addition of 0.2% (w/v) galactose or 2% (w/v) glucose for control cultures.
After 2 h, cells were pelleted, resuspended and measured for GFP fluorescence
intensity using a Becton Dickinson FACSCalibur flow cytometer. For northern
blot analysis, total RNA was isolated, transferred to nylon membrane and
probed as described
48
. Aliquots (3 µg) of total RNA were resolved by agarose-
formaldehyde gel electrophoresis. A GFP probe was generated by BamHI and
NcoI digest of GFP-3xSRE
+
plasmid DNA. SCR1 probe was used as loading
control
49
. For time-course analysis, yeast cells were grown to mid-log phase,
GFP expression was induced by the addition of 2% (w/v) galactose and after
10 min transcription was terminated by the addition of 2% (w/v) glucose.
Aliquots (10 ml) of yeast culture were taken at the indicated time points,
immediately pelleted and frozen on dry ice. RNA was extracted as described
above and 6 µg aliquots of total RNA were resolved by agarose-formaldehyde
gel electrophoresis.
ACKNOWLEDGMENTS
We thank J. Glover and M. Tyers for plasmids p413–GFP and FLAG–VTS1,
respectively. We also thank D. Durocher and R. Collins for assistance with assay
development and A. Willems for relating the similarity between the SSR1 domain
of Smaug and the dimerization domain of βTRCP. We are grateful to P.S.
© 2003 Nature Publishing Group
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ARTICLES
NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 8 AUGUST 2003 621
Metalnikov and P. O’Donnel for assistance with mass spectrometry. C.A.S is
supported by a Canadian Institutes of Health Research (CIHR) scholarship. F.S. is
a Research Scientist of the National Cancer Institute of Canada (NCIC). This work
was supported by operating grants from the CIHR and the NCIC with funds from
the Terry Fox Run.
Note: Supplementary information is available on the Nature Structural Biology website.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 7 March; accepted 24 June 2003
Published online 13 July 2003; doi:10.1038/nsb956
1. Jousset, C. et al. A domain of TEL conserved in a subset of ETS proteins defines a
specific oligomerization interface essential to the mitogenic properties of the TEL-
PDGFR β oncoprotein. EMBO J. 16, 69–82 (1997).
2. Kyba, M. & Brock, H.W. The SAM domain of polyhomeotic, RAE28, and scm medi-
ates specific interactions through conserved residues. Dev. Genet. 22, 74–84
(1998).
3. Barr, M.M., Tu, H., Van Aelst, L. & Wigler, M. Identification of Ste4 as a potential reg-
ulator of Byr2 in the sexual response pathway of Schizosaccharomyces pombe. Mol.
Cell. Biol. 16, 5597–5603 (1996).
4. Wu, C., Leberer, E., Thomas, D.Y. & Whiteway, M. Functional characterization of the
interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae. Mol. Biol.
Cell 10, 2425–2440 (1999).
5. Golub, T.R., Barker, G.F., Lovett, M. & Gilliland, D.G. Fusion of PDGF receptor beta to
a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromo-
somal translocation. Cell 77, 307–316 (1994).
6. Golub, T.R. et al. Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in
acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 92, 4917–4921 (1995).
7. Golub, T.R., Barker, G.F., Stegmaier, K. & Gilliland, D.G. Involvement of the TEL gene
in hematologic malignancy by diverse molecular genetic mechanisms. Curr. Top.
Microbiol. Immunol. 211, 279–288 (1996).
8. Lacronique, V. et al. A TEL-JAK2 fusion protein with constitutive kinase activity in
human leukemia. Science 278, 1309–1312 (1997).
9. Slupsky, C.M. et al. Structure of the Ets-1 pointed domain and mitogen-activated pro-
tein kinase phosphorylation site. Proc. Natl. Acad. Sci. USA 95, 12129–12134
(1998).
10. Chi, S.W., Ayed, A. & Arrowsmith, C.H. Solution structure of a conserved C-terminal
domain of p73 with structural homology to the SAM domain. EMBO J. 18,
4438–4445 (1999).
11. Thanos, C.D. et al. Monomeric structure of the human EphB2 sterile α motif domain.
J. Biol. Chem. 274, 37301–37306 (1999).
12. Wang, W.K. et al. Structure of the C-terminal sterile α-motif (SAM) domain of human
p73 α. Acta. Crystallogr. D. 57, 545–551 (2001).
13. Stapleton, D., Balan, I., Pawson, T. & Sicheri, F. The crystal structure of an Eph
receptor SAM domain reveals a mechanism for modular dimerization. Nat. Struct.
Biol. 6, 44–49 (1999).
14. Thanos, C.D., Goodwill, K.E. & Bowie, J.U. Oligomeric structure of the human EphB2
receptor SAM domain. Science 283, 833–836 (1999).
15. Smalla, M. et al. Solution structure of the receptor tyrosine kinase EphB2 SAM
domain and identification of two distinct homotypic interaction sites. Protein Sci. 8,
1954–1961 (1999).
16. Kim, C.A. et al. Polymerization of the SAM domain of TEL in leukemogenesis and
transcriptional repression. EMBO J. 20, 4173–4182 (2001).
17. Kim, C.A., Gingery, M., Pilpa, R.M. & Bowie, J.U. The SAM domain of polyhomeotic
forms a helical polymer. Nat. Struct. Biol. 9, 453–457 (2002).
18. Smibert, C.A., Lie, Y.S., Shillinglaw, W., Henzel, W.J. & Macdonald, P.M. Smaug, a
novel and conserved protein, contributes to repression of nanos mRNA translation in
vitro. RNA 5, 1535–1547 (1999).
19. Dahanukar, A., Walker, J.A. & Wharton, R.P. Smaug, a novel RNA-binding protein that
operates a translational switch in Drosophila. Mol. Cell 4, 209–218 (1999).
20. Wang, C. & Lehmann, R. Nanos is the localized posterior determinant in Drosophila.
Cell 66, 637–647 (1991).
21. Gavis, E.R. & Lehmann, R. Localization of nanos RNA controls embryonic polarity.
Cell 71, 301–313 (1992).
22. Wang, C., Dickinson, L.K. & Lehmann, R. Genetics of nanos localization in
Drosophila. Dev. Dyn. 199, 103–115 (1994).
23. Bergsten, S.E. & Gavis, E.R. Role for mRNA localization in translational activation
but not spatial restriction of nanos RNA. Development 126, 659–669 (1999).
24. Gavis, E.R. & Lehmann, R. Translational regulation of nanos by RNA localization.
Nature 369, 315–318 (1994).
25. Smibert, C.A., Wilson, J.E., Kerr, K. & Macdonald, P.M. smaug protein represses
translation of unlocalized nanos mRNA in the Drosophila embryo. Genes Dev. 10,
2600–2609 (1996).
26. LeTilly, V. & Royer, C.A. Fluorescence anisotropy assays implicate protein-protein
interactions in regulating trp repressor DNA binding. Biochemistry 32, 7753–7758
(1993).
27. Dilcher, M., Kohler, B. & von Mollard, G.F. Genetic interactions with the yeast
Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6. J. Biol. Chem. 276,
34537–34544 (2001).
28. Crucs, S., Chatterjee, S. & Gavis, E.R. Overlapping but distinct RNA elements control
repression and activation of nanos translation. Mol. Cell 5, 457–467 (2000).
29. McCarthy, J.E. Posttranscriptional control of gene expression in yeast. Microbiol. Mol.
Biol. Rev. 62, 1492–1553 (1998).
30. Tucker, M. et al. The transcription factor associated Ccr4 and Caf1 proteins are com-
ponents of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae.
Cell 104, 377–386 (2001).
31. Tucker, M., Staples, R.R., Valencia-Sanchez, M.A., Muhlrad, D. & Parker, R. Ccr4p is
the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in
Saccharomyces cerevisiae. EMBO J. 21, 1427–1436 (2002).
32. Brown, C.E., Tarun, S.Z. Jr., Boeck, R. & Sachs, A.B. PAN3 encodes a subunit of the
Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae. Mol. Cell. Biol. 16,
5744–5753 (1996).
33. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. Crystallographic analysis of
the recognition of a nuclear localization signal by the nuclear import factor karyo-
pherin α. Cell 94, 193–204 (1998).
34. Huber, A.H. & Weis, W.I. The structure of the β-catenin/E-cadherin complex and the
molecular basis of diverse ligand recognition by β-catenin. Cell 105, 391-402
(2001).
35. Edwards, T.A., Pyle, S.E., Wharton, R.P. & Aggarwal, A.K. Structure of Pumilio
reveals similarity between RNA and peptide binding motifs. Cell 105, 281–289
(2001).
36. Wang, X., McLachlan, J., Zamore, P.D. & Hall, T.M. Modular recognition of RNA by a
human pumilio-homology domain. Cell 110, 501–12 (2002).
37. Jacobson, A. & Peltz, S.W. Interrelationships of the pathways of mRNA decay and
translation in eukaryotic cells. Annu. Rev. Biochem. 65, 693–739 (1996).
38. Gavis, E.R., Lunsford, L., Bergsten, S.E. & Lehmann, R. A conserved 90 nucleotide
element mediates translational repression of nanos RNA. Development 122,
2791–2800 (1996).
39. Suzuki, H. et al. Homodimer of two F-box proteins betaTrCP1 or betaTrCP2 binds to
IκBα for signal-dependent ubiquitination. J. Biol. Chem. 275, 2877–2884
(2000).
40. Kominami, K., Ochotorena, I. & Toda, T. Two F-box/WD-repeat proteins Pop1 and
Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast
SCF (Skp1-Cullin- 1-F-box) ubiquitin ligase. Genes Cells 3, 721–735 (1998).
41. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. The
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment
aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).
42. Page, R.D.M. TREEVIEW: an application to display phylogenetic trees on personal
computers. Comput. Appl. Biosci. 12, 357 (1996).
43. Ponting, C.P., Schultz, J., Milpetz, F. & Bork, P. SMART: identification and annotation
of domains from signalling and extracellular protein sequences. Nucleic Acids Res.
27, 229–232 (1999).
44. Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment
for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).
45. Carson, M. Ribbons. Methods Enzymol. 277, 493–505 (1997).
46. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: insights from the
interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296
(1991).
47. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cere-
visiae by mass spectrometry. Nature 415, 180–183 (2002).
48. Tyers, M. et al. Characterization of G1 and mitotic cyclins of budding yeast. Cold
Spring Harb. Symp. Quant. Biol. 56, 21–32 (1991).
49. Bonnerot, C., Boeck, R. & Lapeyre, B. The two proteins Pat1p (Mrt1p) and Spb8p
interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p.
Mol. Cell. Biol. 20, 5939–5946 (2000).
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