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J. Biol. Chem., Vol. 276, Issue 33, 30803-30811, August 17, 2001
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,§,
From the Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, and Departments of Oncology, Medicine, Microbiology and Immunology, McGill University, Montreal, Quebec H3T 1E2, Canada
Received for publication, March 13, 2001, and in revised form, June 4, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Sam68 is an RNA-binding protein that contains a
heterogeneous nuclear ribonucleoprotein K homology domain embedded in a
larger RNA binding domain called the GSG (GRP33,
Sam68, GLD-1) domain. This family of
proteins is often referred to as the STAR (signal transduction and activators of RNA
metabolism) proteins. It is not known whether Sam68 is a general
nonspecific RNA-binding protein or whether it recognizes specific
response elements in mRNAs with high affinity. Sam68 has
been shown to bind homopolymeric RNA and a synthetic RNA sequence
called G8-5 that has a core UAAA motif. Here we performed a structure
function analysis of Sam68 and identified two arginine glycine
(RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG
domain. In addition, by using chimeric proteins between Sam68 and
QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the
GSG domain of QKI-7, another STAR protein. Furthermore, that minimal
sequence can also give QKI-7 the ability (as Sam68) to functionally
substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our
studies suggest that neighboring RG-rich sequences may impose
nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding
activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein
when tyrosine phosphorylated.
Sam68 (Src substrate associated during
mitosis of 68 kDa)1 is a substrate for
tyrosine kinases including Src family kinases p60src (1-4),
p59fyn (5), p56lck (6), BRK/SIK (7), and ZAP70 (8).
Sam68 has been shown to bind numerous Src homology 3, Src homology 2, and WW domain-containing proteins, leading several groups to suggest
that Sam68 may be an adaptor protein for tyrosine kinases (5, 9). Sam68
is an RNA-binding protein that contains a KH domain embedded in a larger domain of ~200 amino acids, the GSG (GRP33,
Sam68, and GLD-1) domain. Sam68 has been shown to
bind homopolymeric RNA poly(U) and poly(A) (2, 10). The tyrosine
phosphorylation of Sam68 by p59fyn severely inhibits its
ability to bind poly(U)-Sepharose (11). Sam68 has also been shown to
bind synthetic RNA sequences with a core UAAA with high affinity (12).
The function of Sam68 is unknown, but recent findings suggest that it
may be involved in the regulation of splicing and/or RNA transport
(13-16).
The GSG domain is an evolutionarily conserved protein module initially
identified by aligning the first three members of this family (17, 18).
In addition to the KH domain, the GSG domain contains ~75 amino acids
N-terminal and ~25 amino acids C-terminal to the KH domain called the
NK (N-terminal of KH) and CK
(C-terminal of KH) regions, respectively
(schematically represented in Fig. 1). Several properties have been
ascribed to the GSG domain including RNA binding (10, 12, 18-21),
self-association (10, 18, 19, 22), heterodimerization (10, 20, 23), and
protein localization (13).
GSG domain-containing proteins are called STAR proteins for
signal transduction and activators of
RNA metabolism (24, 25), and Sam68 is the prototype because
of its links to signaling proteins (5, 9). The GSG domain is found in a
rapidly growing family of RNA-binding proteins (25). Genetic and
biochemical evidence has demonstrated that STAR proteins are involved
in many essential processes such as splicing (26, 27), tumorigenesis
(17, 28), apoptosis (18, 19, 29), cell cycle progression (30), translation (31, 32), and development (17, 22, 33, 34).
The physiological importance of the GSG domain is demonstrated by the
fact that many genetic mutations that result in growth or developmental
defects have been identified in this protein module. In the nematode
Caenorhabditis elegans, the GSG protein GLD-1 functions as a
tumor suppressor that is required for normal oocyte development (35,
36). Thirty-two gld-1 mutations have been identified
that fall into six phenotypic classes (17). In mice, a missense
mutation in the quaking gene (qk) has been identified (24) that is known to be embryonic-lethal (37). This
mutation, altering glutamic acid 48 to glycine (24), occurs in the NK
region of the GSG domain and has been shown to prevent QKI dimerization
(19). In Drosophila melanogaster, HOW plays a
critical role in skeletal muscle development, because weak alleles result in the "held-out-wings"
phenotype (33, 34).
The phenotype of the quaking viable and lethal
mice suggests that the QKI proteins are involved in myelination and
early embryogenesis (24, 38). The mouse qk gene expresses at
least five alternatively spliced mRNAs including QKI-5, QKI-6, and
QKI-7 that differ in their C-terminal 30 amino acids (24). The
quaking viable mutation, which prevents the expression of
QKI-6 and QKI-7 isoforms in oligodendrocytes (39), severely impairs
myelination, and as a result the mice develop a characteristic tremor
after birth (40). The specific RNA targets of QKI have not been
identified, but the sequence identity between C. elegans
GLD-1 and mouse QKI proteins has led Goodwin and co-workers (41) to
examine whether the introduction of QKI-6 in C. elegans can
regulate the specific RNA target of GLD-1. Indeed QKI-6, similar to
GLD-1, translationally suppressed the expression of TRA-2 and bound
with high affinity to the "tra-2 and GLI
elements" (TGEs) (41).
Previously we have shown that the Sam68 GSG domain in addition to 50 amino acids at its C terminus are necessary and sufficient for RNA
binding (10). To examine the role of the C-terminal amino acids in RNA
binding, we generated chimeric proteins between two STAR proteins,
Sam68 and QKI. Because the primary amino acid sequence of QKI-5, -6, and -7 isoforms are identical except for the last 6-30 amino acids
(depending on the isoform) and are predicted to have identical RNA
binding specificity, we chose QKI-7 for our analysis. Here we
identified two small regions harboring arginine-glycine repeats in
Sam68 that can confer nonspecific RNA binding activity to an adjacent
GSG domain. In addition, a novel Sam68 dimerization region has been
identified in its C-terminal sequences.
DNA Constructions--
The constructs encoding Myc-QKI-7,
Myc-QKI-7:E48G, Myc-Sam68, and Myc-Sam68
The plasmids encoding Myc-Q(GSG)-S, Myc-Q(GSG)-S
The constructs encoding Myc-S
The construct encoding the G8-5 RNA sequence (12), 5'-GGG UGA CAC ACU
AGC UAU AGC AUU AAA AGA CCG AGC AAG U-3' (the UAAA motif is
underlined), was generated by annealing two oligonucleotides (5'-GCC
GAA TTC GGG TGA CAC ACT AGC TAT AGC ATT A-3' and 5'-AGC TCT AGA CTT GCT CGG TCT TTT AAT GCT ATA GCT-3') and filling
in the ends with the Klenow fragment of DNA polymerase I. This DNA fragment was digested with EcoRI and XbaI (the
restriction sites are underlined) and subcloned into pBluescript SK(+).
The plasmids encoding the tra-2 3' UTR and a deletion mutant
of tra-2 3' UTR, (
The identities of all the above plasmid constructs were verified by
dideoxynucleotide sequencing.
Protein Expression and Analysis--
Proteins were expressed in
HeLa cells, using the vaccinia virus T7 expression system as described
previously (5). HeLa cells were lysed in lysis buffer (1% Triton
X-100, 150 mM NaCl, and 20 mM Tris-HCl (pH
8.0)), 50 mM NaF, 100 mM sodium vanadate, 0.01% phenylmethanesulfonyl fluoride, 1 µg of aprotinin/ml, and 1 µg of leupeptin/ml), and the cellular debris and nuclei were removed by centrifugation. For immunoprecipitation, the supernatant was
incubated on ice with the specified antibody for 1 h. Then 20 µl
of a 50% protein A-Sepharose slurry was added and incubated at 4 °C
for 30 min with constant end-over-end mixing. The beads were washed
twice with lysis buffer and once with PBS. Protein samples were
analyzed on SDS-polyacrylamide gels and transferred to nitrocellulose
membranes. Immunoblotting was performed using the anti-Myc (9E10),
anti-hemagglutinin (HA), or anti-p59fyn antibodies. The rabbit
anti-p59fyn antibody was provided kindly by André
Veillette (Institut de Recherche Clinique de Montréal,
Université de Montréal, McGill University). The
designated primary antibody was followed by goat anti-mouse or goat
anti-rabbit antibodies conjugated to horse radish peroxidase (ICN), and
chemiluminescence was used for protein detection (DuPont).
In Vitro Transcription--
32P-labeled G8-5 RNA
and tra-2 3'-UTR RNA were transcribed in vitro
with the T7 RNA polymerase following the protocols recommended by the
manufacturer (Promega). After in vitro transcription, the template DNA was digested with DNase I (Promega), and the RNA was
extracted with phenol-chloroform, precipitated with ethanol, and
resuspended in diethyl pyrocarbonate-treated water at a concentration of 106 cpm/µl.
RNA Binding and Functional Assays--
For the poly(U) binding
assay, Myc-tagged proteins expressed in HeLa cells were incubated at
4 °C for 30 min with poly(U)-Sepharose beads or control Sepharose
beads in lysis buffer supplemented with 2 mg/ml heparin. The bound
proteins were separated by SDS-PAGE, transferred to nitrocellulose, and
immunoblotted with anti-Myc antibodies. For G8-5 or tra-2
3'-UTR RNA binding, Myc-tagged proteins expressed in HeLa cells were
immunoprecipitated with an anti-Myc antibody or mouse IgG (control),
and the immunoprecipitates were incubated at 4 °C for 30 min with 1 µl (106 cpm) of 32P-labeled RNA in lysis
buffer supplemented with 2 mg/ml heparin. The beads were washed twice
with lysis buffer and once with PBS, and the bound radioactivity was
quantitated by scintillation counting. To verify the identity of the
radiolabeled RNA bound to beads, the bound RNA was eluted with sample
buffer and analyzed with nondenaturing polyacrylamide gel
electrophoresis and autoradiography. To verify protein expression, the
immunoprecipitates were analyzed by immunoblotting with anti-Myc
antibody. REV assays were performed as described previously
(7).
STAR proteins contain a GSG domain, which is a tripartite protein
module containing from N to C terminus the NK region, the KH domain,
and the CK region (Fig. 1B).
The Sam68 KH domain is necessary for RNA binding because its deletion
prevents RNA binding (10, 12, 30). To investigate whether the RNA
binding specificity of the STAR proteins resides only in the KH domain
or whether neighboring regions can regulate RNA binding, we constructed
chimeric proteins between QKI-7 and Sam68. These two proteins show a
high degree of homology in their GSG domain (Fig. 1A) but
possess distinct RNA binding specificities. Sam68 has been shown to
bind homopolymeric RNA poly(U) and poly(A) (2, 10) as well as a
synthetic RNA (G8-5) amplified by using systematic evolution of
ligands by exponential enrichment (12). The QKI proteins bind
C. elegans GLD-1 target, tra-2 (41), but not
homopolymeric RNA (10). Chimeric proteins were generated between Sam68
and QKI-7 in such a way that the CK region and the C terminus of one
protein was replaced by the corresponding region of the other protein
(Fig. 1B). Q-S (QKI-7-Sam68 chimeric protein) contains the
NK region and the KH domain of QKI-7 and the CK region and the C
terminus of Sam68 (Fig. 1B). S-Q (Sam68-QKI-7 chimeric
protein) contains the Sam68 N-terminal portion, the NK region and the
Sam68 KH domain, and the QKI-7 C terminus including the CK region.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-67 (now renamed
SN) were described previously (5, 10, 19). Myc-S-Q encodes a
chimeric protein containing the N-terminal region of Sam68 and the
C-terminal region of QKI-7. The DNA sequence encoding the C-terminal
180 amino acids of Myc-Sam681-67 was removed by restriction
endonuclease digestion using EcoRV and KpnI and
replaced with a sequence encoding the C-terminal 136 amino acids of
QKI-7. The QKI-7 fragment was generated with polymerase chain reaction
(PCR) using Myc-QKI-7 as the DNA template and the universal reverse
primer and 5'-GAC GAT ATC AAG AAG ATG CAG CTG ATG-3' (the
EcoRV site is underlined) as oligonucleotides. The chimeric
protein Myc-Q-S contains the N-terminal region of QKI-7 and the
C-terminal region of Sam68. The plasmid expressing this protein was
constructed as follows: a DNA fragment encoding the C-terminal 188 amino acids of Sam68 was generated with PCR using Myc-Sam68 as the DNA
template, 5'-CCT GGT ACC AGA TAT GAT GGA T-3' as the
forward primer, and universal reverse primer as the reverse primer, the
fragment was digested with KpnI (the restriction site is
underlined) and subcloned into Myc-QKI-7, the C-terminal 145 amino
acids of which had been removed with KpnI digestion. Myc-Q-S:E
G was constructed using the same strategy as that of Myc-Q-S except that Myc-QKI-7:E48G, instead of Myc-QKI-7, was used to
subclone the Sam68 DNA fragment. The construct encoding Myc-Q-S:AN
was generated by inverse PCR using Myc-Q-S as the DNA template
and 5'-ATT CAA CTT GAA GCA GAA ACG GGA-3' and
5'-ATT TGT AAG TCC TCT AGG TCC AAG-3' as primers
(underlined nucleotides denote changes introduced). The Myc-Q-S and
Myc-S-Q C-terminal deletion constructs, Myc-Q-S330, Myc-Q-S294,
Myc-S-Q284, and Myc-S-Q205, were generated with PCR using Myc-Q-S and
Myc-S-Q as the DNA templates, respectively. The T7 promoter primer was
used as the forward primer and the following oligonucleotides were used
as reverse primers: 5'-AGG AAT TCA TGG CAC CCC TCG AGT CAC
A-3' (for Myc-Q-S330), 5'-TAG AAT TCA GGC AGC TCC TCG TCC
TCT CAC-3' (for Myc-Q-S294), 5'-AGG GAA TTC AGA TTA ACC CAG
CTT CAG GCC-3' (for Myc-S-Q284), and 5'-ATG GAA TTC TAT CTG
TAG GTG CCA TTC AG-3' (for Myc-S-Q205). The amplified DNA fragments
were digested with EcoRI and subcloned into Myc-Bluescript
KS(+) (5, 10).
4RG, and
Myc-Q(GSG)-S6RG were constructed by a two-step subcloning strategy. DNA fragments encoding the C-terminal regions of Sam68 were amplified by PCR with universal reverse primer and 5'-GTG GTC GAC GGG
TAT CTG TGA GAG GAC-3' for Myc-Q(GSG)-S, 5'-GGA GTC GAC CCT
CCT CCT CCA CCT GT-3' for Myc-Q(GSG)-S4RG or 5'-GTG GTC
GAC CAC CTA GAG GAG CTT-3' for Myc-Q(GSG)-S6RG as
primers. The fragments were digested with SalI (the
restriction site is underlined) and KpnI (the site is in the
vector) and inserted in the same sites of pBluescript KS(+). The
resulting plasmids were then used to subclone the DNA fragments
encoding Myc-tagged N-terminal regions of QKI-7. The Myc-QKI-7
fragments were generated by PCR using Myc-QKI-7 as template, T7
promoter primer as the forward primer, and the following
oligonucleotides as reverse primers: 5'-CTG GTC GAC TAA TGT
TGG CGT CTC TGT-3' for Myc-Q(GSG)-S, 5'-AGT GTC GAC AAG AGA
AAA GGC AAG GGC-3' for Myc-Q(GSG)-S4RG, and 5'-CAG GTC
GAC GCC CAG TGA TGA TCC TTG-3' for Myc-Q(GSG)-S6RG. These
fragments were digested with BamHI (the site is in the
vector) and SalI (the site is underlined) and subcloned in
the corresponding Sam68-pBluescript plasmids described above. The
construct encoding Myc-Q(GSG)-S11RG was also generated in two steps.
A PCR fragment encoding the Myc-epitope tag and the N-terminal 256 amino acids of QKI-7 was first subcloned in the BamHI and
SalI sites of pBluescript KS(+), and the XhoI fragment (C-terminal 112 amino acids) of Sam68 was then inserted in the
SalI site of the resulting plasmid. To generate the
Myc-QKI:1-256 fragment, Myc-QKI-7 was used as DNA template and T7
promoter primer and 5'-GTA GTC GAC TGA TCA AAG GCA TTA-3'
as primers (the SalI site is underlined). Myc-QKI-5RG is a
chimeric protein in which a Sam68 sequence harboring five RG repeats is
introduced in QKI-7. The plasmid encoding this protein was generated as
follows: a PCR fragment encoding the C-terminal 65 amino acids of QKI-7
was first inserted in the HindIII and KpnI sites
of pBluescript KS(+), and a second PCR fragment containing coding
sequences for the Myc tag, QKI-7 amino acids 1-231 and Sam68 amino
acids 308-333, was then subcloned in the BamHI and
HindIII sites of the resulting plasmid. The first PCR
fragment was generated using Myc-QKI-7 as template and universal
reverse primer and 5'-AGA AAG CTT TCA TGC CAA ACG GAA
CTC-3' as primers. The second PCR fragment was generated using
Myc-Q(GSG)-S6RG as template and T7 promoter primer and 5'-GTG
AAG CTT GCA CCC CTC GAG TCA CAG-3' as primers (the HindIII site is underlined).
N:315RGAS, Myc-SN:320RGAS,
Myc-SN:325RGAS, Myc-SN:315,325RGAS, Myc-SN:3RG,
Myc-Sam68:3RG, Myc-Sam68RA:10,13,17, Myc-Sam68RA:43,45, and
Myc-Sam68RA:52,56 were all generated by inverse PCR. The
DNA templates used were Myc-SN (for Myc-SN:3RG and
-SN:RGAS constructs except Myc-SN:315,325RGAS), Myc-SN:315RGAS (for Myc-SN:315,325RGAS), Myc-Sam68 (for
Myc-Sam68:3RG), and Myc-Sam68:3RG (for Myc-Sam68RA
constructs), and the primer pairs were: 5'-AGC
ACC CCA GTG AGA GGC TCC-3' and 5'-AGC AAC CAA AGC TCC TCT
AGG-3' (for Myc-SN:315RGAS), 5'-AGC TCC ATC ACC AGA
GGT G-3' and 5'-AGC CAC TGG GGT TCC ACG AAC-3' (for
Myc-SN:320RGAS), 5'-AGC GCC ACT GTG ACT
CGA GGG-3' and 5'-AGC GGT GAT GGA GCC TCT CAC-3' (for
Myc-SN:325RGAS and -SN:315,325RGAS),
5'-AGC GCC ACT GTG ACT CGA GGG-3' and
5'-AGC AAC CAA AGC TCC TCT AGG-3' (for Myc-SN:3RG and
-Sam68:3RG), 5'-AGC TCG GGC GCC AGC TGC TCC AAG GAC CCG-3' and 5'-AGC GGT GAG GGC CGA GGC
AGG ATC GTC CCG-3' (for Myc-Sam68RA:10, 13, 17),
5'-AGC GGG GGA GGT GGG CCC AGA-3' and
5'-AGC GGG CGC GTG AGG AAG CGG CGA CGG-3' (for
Myc-Sam68RA:43,45), and 5'-AGC GGC GCT
GCG GCC TCG CCCGCC ACC CAG-3' and 5'-AGC GGG CCC ACC TCC CCC TCC-3' (for Myc-Sam68RA:52,56). Underlined
nucleotides denote changes introduced.
108) 3' UTR, were kindly provided by Tim
Schedl (Washington University).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of protein
constructs. A, the amino acid sequence homology between
Sam68 and QKI-7 is shown. An asterisk denotes identity,
two dots are representative of semi-conservative amino
acids, and a single dot signifies lower conservation.
B, the QKI-7 protein sequence is shown in black,
and the Sam68 sequence is shown in white. The GSG domain
consists of a KH domain flanked by the NK and CK regions. The chimeric
protein Q-S contains the N-terminal portion of QKI-7 and the C-terminal
portion of Sam68. Q-S:E G and Q-S:AN are identical to Q-S, except
that the lethal point mutation E48G or a point mutation (A110N) that
inactivates the KH domain (equivalent to the fragile X syndrome protein
I304N) was introduced in the GSG domain (indicated by a vertical
line). Q-S330 and Q-S294 are C-terminal deletion mutants of Q-S,
truncating the proteins at Sam68 amino acids 330 and 294. The chimeric
protein S-Q contains the N-terminal portion of SN and the C-terminal
portion of QKI-7. S-Q205 and S-Q285 are truncated proteins of S-Q,
deleting the QKI-7 amino acids at 205 and 285, respectively.
The C-terminal Portion of Sam68 Confers Poly(U) Binding to
QKI-7--
To examine the RNA binding specificity of these chimeric
proteins, Myc-tagged Q-S and S-Q were expressed in HeLa cells and tested for their ability to bind poly(U)-Sepharose. Q-S bound poly(U)-Sepharose, whereas S-Q did not (Fig.
2A, lanes 9 and
12, upper panel). This difference in
binding was also observed if the Q-S and S-Q proteins were incubated
together with the same poly(U)-Sepharose beads, hence eliminating the
possibility of a recovery problem (Fig. 2A, lanes
22-24). As positive and negative controls, respectively,
Sam681-67 (herein renamed SN, Fig. 1B) bound poly(U)
and QKI-7 did not (Fig. 2A, upper panel,
lanes 6 and 3, respectively). We used SN in
this assay because it has been shown to bind poly(U) as well as
full-length Sam68, and its poly(U) binding activity is regulated by
p59fyn (11). We have shown previously that the introduction of
QKI-7 glutamic acid 48 to glycine in the NK region prevents QKI-7
dimerization (19). To examine whether dimerization via the QKI-7 NK
domain was required for Q-S to bind poly(U)-Sepharose, we introduced the E48G amino acid substitution in Q-S. Q-S:EG was expressed in
HeLa cells and tested for its ability to bind poly(U)-Sepharose. The
poly(U) binding activity of Q-S:EG was similar to that observed for
Q-S (Fig. 2A, lane 15, upper panel),
suggesting that self-association via the predicted coiled-coil
region in the QKI-7 NK region was not required for poly(U) binding.
Because we have shown previously that Sam68 devoid of its KH domain
does not bind poly(U) (10), these findings demonstrated that sequences
neighboring the KH domain cannot bind poly(U) per se. Thus
the ability of Q-S to bind poly(U) suggested that the Sam68 sequences
conferred to the QKI-7 KH domain the ability to bind
poly(U)-Sepharose.
|
To further delineate the Sam68 portion required to confer poly(U)
binding to the Q-S chimeric protein, we performed C-terminal deletions
resulting in Q-S294 and Q-S330 (Fig. 1B). We have shown previously that in the context of the SN protein, 50 amino acids C-terminal to the GSG domain are required for RNA binding such that
Sam68:330 bound RNA and Sam68:294 did not (10). We examined whether the
Q-S chimeric protein truncated at Sam68 amino acids 294 and 330 bound
poly(U)-Sepharose. The chimeric protein Q-S330 bound poly(U)-Sepharose
and Q-S294 did not (Fig. 2A, lanes 18 and
21, upper panel). These results demonstrated that
Q-S, interestingly, was behaving like Sam68. Thus the minimal Sam68
sequence required to confer poly(U) RNA binding to QKI-7 resided from
Sam68 amino acids 256-330, a region harboring the CK region as well as
an additional 50 amino acids.
The poly(U) binding activity of SN is negatively regulated by
p59fyn (11). Thus we examined the effect of p59fyn on
the poly(U) binding activity of the chimeric proteins. The chimeric
proteins were co-expressed with p59fyn in HeLa cells, and their
poly(U) binding activity was examined. The poly(U) binding activity of
Q-S and Q-S:EG was severely impaired with the co-expression of
p59fyn (Fig. 2A, lanes 12 and
15, lower panel). These results indicated that
the poly(U) binding activity of Q-S and Q-S:EG was regulated by
p59fyn and further demonstrated that Q-S behaved like Sam68.
The poly(U) binding of Q-S330 was not affected by p59fyn (Fig.
2A, lane 18, lower panel). This
finding was expected, because Q-S330 does not contain the
phosphorylation sites for p59fyn that reside in the C terminus
of Sam68 (5). The RNA binding activity of SN was inhibited by the
expression of p59fyn and served as a positive control for the
assay (Fig. 2A, lane 6, lower panel).
The expression of p59fyn was confirmed by immunoblotting an
aliquot of total cell lysate corresponding to Fig. 2A with
anti-p59fyn antibodies (Fig. 2B). Because the C
terminus of Sam68 harbors a regulatory domain that can abrogate RNA
binding when phosphorylated by p59fyn on tyrosine residues
(11), it was conceivable that the C-terminal region of QKI-7 in S-Q may
be inhibiting the ability of the Sam68 KH domain from binding poly(U).
To eliminate this possibility, we made C-terminal deletions in S-Q
(Fig. 1B, S-Q:284, and S-Q:205) and measured their ability
to interact with poly(U)-Sepharose. None of these chimeric proteins
bound poly(U)-Sepharose (Fig. 2C: individually, lanes 1-12;
mixed, lanes 13-15), demonstrating that the C-terminal
region of QKI-7 does not harbor a sequence that inhibits RNA binding.
An isoleucine to asparagine substitution in the second KH domain of the
fragile X mental retardation gene product (FMRP) is sufficient to
severely impair RNA binding (42). The equivalent amino acid
substitution in QKI-7, alanine 110 to asparagine (AN), was
introduced in Q-S to examine the contribution of the QKI-7 KH domain in
the poly(U) binding observed with Q-S. Q-S:AN had impaired poly(U)
binding compared with wild-type Q-S (Fig. 2D), suggesting
that the QKI-7 KH domain is required for Q-S poly(U) binding. These
findings suggested that the C-terminal sequences of Sam68 are able to
confer a new RNA binding activity to the QKI-7 KH domain.
The Sam68 C-terminal Region Confers G8-5 RNA Binding--
The
physiological RNA targets for Sam68 are unknown, but a degenerate RNA
sequence containing a UAAA motif called G8-5 has been identified by
systematic evolution of ligands by exponential enrichment that
binds Sam68 with high affinity (12). Myc-Sam68, -QKI-7, -S-Q, and -Q-S
expressed in HeLa cells were immunoprecipitated with anti-Myc
antibodies or control mouse IgG, and the immunoprecipitates were
incubated with in vitro transcribed 32P-labeled
G8-5 RNA. The immunoprecipitates were washed, and the amount of bound
RNA was quantitated and expressed as counts per minute (Fig.
3A). The radioactivity bound
by Sam68 and Q-S anti-Myc immunoprecipitates was 15-20 times higher
than control immunoprecipitates, whereas there was only a 2-3-fold
difference between anti-Myc and control immunoprecipitates of QKI-7 and
S-Q (Fig. 3A). The bound RNAs were analyzed with
nondenaturing polyacrylamide electrophoresis and visualized by
autoradiography to verify that the radioactivity correlated with
32P-labeled G8-5. The G8-5 RNA was observed in Sam68 and
Q-S Myc immunoprecipitates (Fig. 3B), confirming that Sam68
and Q-S bound G8-5. The absence of G8-5 binding with QKI-7 and S-Q
was not caused by a lower expression of these proteins, because
anti-Myc immunoblotting of the immunoprecipitates showed comparable
expression of the Myc-tagged proteins (Fig. 3C). These data
are consistent with the poly(U) binding results shown in Fig. 2,
confirming that the Q-S chimeric protein has an RNA binding specificity
similar to Sam68.
|
G8-5 RNA Binding Activity of Sam68 and Q-S Is Regulated by
p59fyn--
We investigated whether the ability of Sam68
and Q-S to bind G8-5 was regulated by p59fyn. Although G8-5
is a known high affinity RNA target for Sam68, it is not known whether
G8-5 RNA binding is regulated by tyrosine phosphorylation. Myc-Sam68,
-SN, and -Q-S were transfected in HeLa cells with or without
p59fyn, the cells were lysed, and the lysates were
immunoprecipitated with anti-Myc or control IgG antibodies. The
immunoprecipitated proteins were subsequently incubated with
32P-labeled G8-5 RNA, and the beads were washed, counted
in a scintillation counter, and expressed in counts per minute. The
G8-5 binding activity of full-length Sam68, SN, and Q-S was
inhibited by co-expression of p59fyn (Fig.
4A). The expression of
p59fyn nearly abolished G8-5 binding to SN, whereas G8-5
binding to Sam68 and Q-S was reduced by ~50% (Fig. 4A).
These findings suggested that the Sam68 N-terminal 67 amino acids
regulate the ability of Src kinases to negatively abrogate RNA binding.
Equivalent Myc and p59fyn expression were observed in the
different samples (Fig. 4, B and C). These data
further demonstrated that Q-S behaved like Sam68.
|
The RG Repeats in Sam68 Are Necessary for Poly(U) Binding--
The
minimal region of Sam68 required to confer poly(U) RNA binding to QKI-7
resided in Sam68 amino acids 256-330, which included the CK region
(Fig. 1B, Q-S330). To verify whether the Sam68 sequence harboring the CK region or the RG repeats within the additional 50 amino acids was responsible for the new specificity of the Q-S chimera,
a new chimeric protein was constructed that extended the QKI-7
sequences to include its CK region. This chimeric protein named
Q(GSG)-S, which now contained the entire QKI-7 GSG domain, was tested
for its ability to bind poly(U)-Sepharose. Q(GSG)-S retained the
ability to bind poly(U) to the same extent as SN or Q-S (Fig.
5A). Thus the sequences
C-terminal of the Sam68 GSG domain, and not the Sam68 CK region, were
capable of conferring poly(U) binding to the QKI-7 GSG domain. The
minimal region essential to confer poly(U) binding specificity to QKI-7
was mapped by engineering chimeric proteins where the junction between
QKI-7 and Sam68 sequences was gradually displaced toward the C
terminus. The chimeric proteins were named according to the number of
RG repeats that were deleted (Fig. 5A: Q(GSG)-S4RG,
6RG, and 11RG). These chimeras were expressed in HeLa cells and
tested for their ability to bind poly(U)-Sepharose. The deletion of
four or six RG repeats had little or no effect on the ability of the
QKI-7-Sam68 chimeras to bind poly(U)-Sepharose (Fig. 5A:
Q(GSG)-S4RG and 6RG). In contrast, a larger truncation deleting
11 RG repeats of the Sam68 sequence completely abolished poly(U)
binding (Fig. 5A: Q(GSG)-S11RG). The minimal Sam68
sequence capable of changing the RNA binding specificity of the QKI-7
GSG domain was located between amino acids 308-333, which harbors five
RG repeats. This 26-amino acid sequence from Sam68 was introduced at a
similar position in QKI-7. The plasmid expressing this QKI-5RG chimeric
protein was transfected in HeLa cells and examined for its ability to
bind poly(U)-Sepharose. The chimeric protein QKI-5RG bound
poly(U)-Sepharose (Fig. 5A). These findings demonstrate that
the Sam68 26 amino acids spanning amino acids 308-333 are sufficient
for conferring homopolymeric RNA binding to the QKI-7 GSG domain. As a
control for loading and recovery, a representative poly(U) binding
reaction with Myc-tagged QKI-7 was reimmunoblotted using anti-Sam68
antibodies, confirming that endogenous Sam68 bound poly(U) and not the
epitope-tagged Myc-QKI-7 (Fig. 5B, upper half).
|
Three of the five RG repeats in the SN sequences spanning amino
acids 308-333 were replaced with alanine and serine residues, respectively. The SN RGAS mutant proteins were expressed in HeLa
cells, and their capacity to bind poly(U) was examined (Fig. 6). If the RG repeats participate in
conferring homopolymeric RNA binding to the Sam68 GSG domain, the
removal by amino acid substitution or deletion should prevent poly(U)
binding. The substitution of the individual RGs at position 315, 320, or 325 had a comparable effect such that poly(U) binding was reduced by
more than 50% (Fig. 6: SN:315RGAS, 320RGAS, and 325RGAS).
When the double substitution of 315 and 325 or the deletion of amino
acids 315-325 was performed, poly(U) binding was completely abrogated
(Fig. 6: 315,325RGAS and SN3RG). These data demonstrate that
the RG repeats at positions 315 and 325 are necessary for Sam68 poly(U) binding.
|
The deletion of amino acids 315-325 was performed next in the context
of the full-length Sam68 protein, and its ability to bind
poly(U)-Sepharose was examined (Fig. 6: Sam683RG). Sam683RG bound
poly(U)-Sepharose with wild-type affinities unlike SN:3RG (Fig.
6). These findings suggested that the N-terminal sequences of Sam68
function in a redundant manner with the residues located between 315 and 325 to confer poly(U) binding to the Sam68 GSG domain. The Sam68
N-terminal 67 amino acids (1, 5) harbor several individual arginine
residues and a motif that matches the consensus of an RGG box, a type
of RNA binding motif (43). To identify the N-terminal sequence required
to confer poly(U) binding to the Sam68 GSG domain, a series of mutant
proteins was engineered in the Sam683RG "background."
Sam683RG was chosen to eliminate any contribution from the
C-terminal 315-325 amino acids. Alteration of arginines 10, 13, and 17 to alanines had a minor effect on poly(U) RNA binding (Fig. 6:
RA:10, 13, 17). In contrast, substitution of arginines at position
43 (just outside the RGG box) and 45 (eliminating the first RGG repeat)
to alanines was sufficient to severely impair the interaction with
poly(U) RNA (Fig. 6: RA:43, 45). Removing the second RGG sequence as well as the adjacent arginine had little or no effect on poly(U) binding (RA:52, 56), suggesting that the first RGG sequence plays a
major role in conferring poly(U) specificity to the Sam68 GSG domain.
The RG Repeats Can Confer QKI-7 the Ability to Substitute for REV
in an HIV RNA Export Assay--
The cellular role of Sam68 is still
unknown, but it has been proposed to be the cellular homologue of REV
in transporting HIV RNA (14). We examined whether the QKI-Sam68
chimeras could functionally substitute for REV in mediating a REV
response element (RRE)-directed reporter gene expression. COS-7 cells
were transfected with an RRE-chloramphenicol acetyltransferase (CAT)
reporter plasmid in the presence of expression vectors encoding REV,
SN, QKI-7, or the chimeric proteins. The presence of REV or SN
induced an approximate 8-fold increase in CAT activity that was not
observed in cells co-transfected with vector alone (pcDNA) or a
mutant of REV(M10) that is RNA binding-defective (Fig.
7). The transfection of Q-S induced an
approximate 6-fold increase in CAT activity consistent with our data
that Q-S binds poly(U)-Sepharose and G8-5. QKI-7 and the S-Q chimeric
protein had no significant activity on the RRE-CAT reporter plasmid
(Fig. 7). The deletion of the three RG repeats in the SN protein
(SN:3RG) abolished its REV-like activity, and the addition of
Sam68 amino acids 308-333 in QKI-7 (QKI-5RG) conferred REV-like
activity to QKI-7. These results demonstrate that the RG-rich sequences
are necessary for poly(U) and G8-5 binding and for Sam68 to
functionally substitute for REV in the transport of RNAs.
|
The C-terminal Portion of Sam68 Harbors a Region That Mediates
Self-association--
STAR proteins have been shown to self-associate
into oligomers (10). We have shown that the Sam68 KH domain and the
QKI-7 NK region are required for self-association (10, 19). The introduction of the E48G mutation in NK region abolishes QKI-7 self-association (19). To investigate the ability of the chimeric proteins to associate with either Sam68 or QKI-7, Myc-Sam68, -QKI-7, -S-Q, -Q-S, -Q-S:EG, and Q-S330 were individually co-expressed with
either HA-tagged Sam68 or QKI-7 in HeLa cells. The transfected cells
were lysed, and the cell lysates were immunoprecipitated with anti-Myc
antibody or control mouse IgG. The immunoprecipitates as well as an
aliquot of the corresponding total cell lysates were separated by
SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-HA
antibodies. The presence of HA-Sam68 or HA-QKI-7 in anti-Myc
immunoprecipitates indicated an association of Myc-tagged proteins with
Sam68 or QKI-7. Sam68 and QKI-7 self-associated (Fig.
8, lanes 3 and 12)
and did not associate with each other (lanes 6 and
9). S-Q associated with Sam68 (lane 15)
consistent with the Sam68 self-association region residing in the KH
domain. The S-Q chimeric protein did not interact with QKI-7, as
predicted (lane 18). Surprisingly, Q-S associated with both
Sam68 and QKI-7 (Fig. 8, lanes 21 and 24). The
association of Q-S with QKI-7 was expected because the chimeric protein
contains the QKI-7 NK region. However, the association with Sam68 was
not predicted and suggests that another multimerization region resides
in the Sam68 C-terminal 188 amino acids. The deletion of the C-terminal
113 amino acids in Q-S (Q-S330) abolished the association with Sam68
(lane 33) but maintained the association with QKI-7
(lane 36). Similar results were obtained when the C-terminal
149 amino acids of Sam68 were deleted from Q-S (Q-S294, data not
shown). The chimeric protein harboring the QKI-7 lethal mutation
Q-S:EG did not associate with QKI-7, but the association with
Sam68 remained intact (lane 30), consistent with the idea
that the NK region of QKI-7 mediates the association with QKI-7. In
summary, the chimeric proteins associated with QKI-7 in a predicted
fashion: if the NK region of QKI-7 was present, there was association
with QKI-7, and if the NK region was absent or if it contained the
lethal point mutation E48G, there was no association with QKI-7. The
association with Sam68 was more complex. Both the known region for
self-association, namely the KH domain, and a newly identified region
located in the C-terminal 113 amino acids were involved in association
with Sam68. Thus the usage of chimeric proteins has permitted the
discovery of a region in the C terminus of Sam68 that is involved in
multimerization that was not observed by deletion and/or mutation
analysis (10).
|
Tra-2 3'-UTR RNA Binding Activity of Sam68, QKI-7, and Chimeric
Proteins--
One question that remained unanswered was whether the
S-Q chimera had gained a QKI-like RNA binding specificity. In the
course of these studies it was shown by Goodwin and co-workers (41) that QKI-7 bound the 3' UTR of C. elegans tra-2. To test
whether the chimeric proteins associated with an RNA target bound by
QKI-7, Myc-tagged Sam68/QKI-7 chimeric proteins were expressed in HeLa cells and immunoprecipitated with anti-Myc or control IgG, and immunoprecipitates were incubated with in vitro transcribed
32P-labeled tra-2 3'-UTR RNA or a mutant RNA
with a deletion of 108 nucleotides (108 3' UTR, Fig.
9C). The amount of bound RNA after several washes was quantitated and expressed as counts per minute. Anti-Myc immunoprecipitates of QKI-7 bound tra-2
(~70,000 cpm, Fig. 9A) but not the 108 mutant
RNA, which is consistent with previous studies (41). In contrast,
Sam68, Q-S, and S-Q had negligible tra2 binding (<1000 cpm,
Fig. 9A). The absence of binding with Sam68, Q-S, and S-Q
was not caused by a lower expression of these proteins, because
anti-Myc immunoblotting of the immunoprecipitates showed comparable
expression of all Myc-tagged proteins (Fig. 9B). The
Q(GSG)-S chimera, which contained an intact QKI-7 GSG domain, bound
very strongly to the tra-2 3'-UTR RNA, to a level comparable
with the wild-type QKI-7 protein (Fig. 9A). These results
demonstrate that the entire QKI-7 GSG was required for tra-2
binding and that the C terminus of Sam68 did not participate or
influence this binding (Fig. 9 and data not shown).
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| |
DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we demonstrate that an RG-rich sequence C-terminal to the Sam68 GSG domain spanning amino acids 308-333 is necessary for poly(U) binding, G8-5 binding, and functionally substituting for REV in the transport of HIV RNAs. Using chimeric proteins, we also demonstrate that these 26 amino acids of Sam68 are sufficient to confer to another unrelated STAR protein, QKI-7, the ability to bind poly(U)-Sepharose and G8-5 and functionally substitute for REV in the transport of HIV RNAs. Because a functional KH domain is required for these three activities of Sam68 (10, 12, 14, 30), it is clear that the RG sequences themselves do not possess intrinsic RNA binding activity. The most likely explanation is that the RG repeats confer to neighboring GSG domains the ability to bind certain RNAs. The collaboration between the RG-rich regions of Sam68 with the GSG domain is reminiscent of a study by Rosbach and co-workers (44) on the GSG domain of BBP/SF1, another STAR protein. BBP/SF1 contains both a GSG domain and a Zn knuckle RNA binding motif. The GSG domain of these proteins is involved in specific recognition of the pre-mRNA branchpoint sequence, but one or more accessory modules is required to achieve efficient binding (44).
The inhibitory effect of p59fyn on SN was more severe than
on full-length Sam68, suggesting that the N-terminal region of Sam68 may contain regulatory sequences. The mouse Sam68 N-terminal 67 amino
acids contain two RGG repeats interspaced by four residues (5) and may
be a bona fide RGG box, a type of RNA binding motif (43).
The deletion of the Sam68 N-terminal 67 amino acids had no effect on
poly(U) and G8-5 RNA binding, suggesting that the RGG repeats do not
play a major role in RNA binding or that other sequences in Sam68
function in a redundant manner. Indeed, RG sequences on either side of
the GSG domain function in a redundant manner to confer poly(U) binding
to the GSG domain. Deletion of the N-terminal RGG boxes or deletion of
amino acids 315-325 had no effect on poly(U) binding. However, a Sam68
protein containing the double deletion/mutation was unable to associate
with poly(U)-Sepharose. The tyrosine phosphorylation of SN by
p59fyn has been shown to abrogate poly(U) binding (11),
suggesting that the phosphorylated C terminus of Sam68 can negatively
regulate the nonspecific RNA binding contributions from RG-rich
sequences located from 315-325. The fact that the full-length protein
was less affected by phosphorylation by p59fyn suggests several
possibilities: 1) the N-terminal RG sequences are not regulated by
p59fyn and may require additional signals such as arginine
methylation (15), and 2) a tyrosine kinase may phosphorylate different
tyrosines on Sam68 that may now regulate the N-terminal RG region. In
summary, our analysis has uncovered a role for the N-terminal RGG
sequences that was not obvious using simple deletion strategies.
The Q-S chimeric protein has lost its ability to interact with a QKI-7-specific RNA target, the 3' UTR of tra-2 (see Fig. 9). Previous studies demonstrated that the GSG domain was the minimal region required for RNA binding and that GSG proteins devoid of a CK region had impaired RNA binding (19, 21). Our results are consistent with this notion, because we show that if the complete GSG domain of QKI-7 is included in the chimeras (Q(GSG)-S) we now observe specific binding to the tra-2 RNA. Moreover, the Q(GSG)-S chimeric protein still displays Sam68-like RNA binding specificity, namely interaction with poly(U) and G8-5 RNAs. Thus the GSG domain is the only RNA binding region in the QKI-7 protein required for specific high affinity RNA binding.
The construction of chimeric QKI-7/Sam68 proteins has also permitted us
to find an oligomerization region located in the Sam68 C-terminal 113 amino acids. Our previous studies demonstrate that the Sam68 GSG domain
(Sam68:103-269) is able to associate with a wild-type Sam68 protein
and that a deletion in the KH domain of Sam68 (Sam68KH) abolished
self-association (10). These data showed that the GSG domain was
necessary and sufficient for self-association and that the Sam68
C-terminal ~200 amino acids in Sam68KH were not sufficient,
without an intact KH domain, to mediate self-association (10). Using
QKI-7/Sam68 chimeras, it is evident that the C-terminal region of Sam68
harbors a region required for self-association. In this situation the
QKI-7 KH domain seems to compensate for the loss of the Sam68 KH
domain. The coiled coil in the NK region of QKI-7 did not participate
in the Sam68 association, because the introduction of the E48G
substitution (Q-S:EG), known to abolish the association with QKI-7
(19), did not affect the association with Sam68. The presence of two
regions in Sam68 that mediate self-association suggests that Sam68 may
be forming head-to-tail multimers and/or that Sam68 is involved in
intramolecular interactions. We have shown previously that the tyrosine
phosphorylation of Sam68 by p59fyn prevents self-association
(10). The mechanism by which tyrosine phosphorylation regulates
self-association is unknown. Now with the discovery of a new region
that resides in the C-terminal tyrosine-rich region of Sam68, it is
possible that the GSG domain associates with a region in the C-terminal
tyrosine region and that the phosphorylation of this region by Src
kinases would interfere with this self-association, which is consistent
with our previous observations (10).
Based on the results presented in this study, we propose the following
model: Sam68 in the resting state would be nonphosphorylated and
exhibit nonspecific RNA binding mediated by the concerted effort of its
GSG domain and its flanking RG-rich regions. The tyrosine
phosphorylation of Sam68 would render it a specific RNA-binding protein. Supporting this model is the fact that poly(U) binding, the
UAAA RNA target G8-5, and REV-like function of Sam68 were identified
by using unphosphorylated Sam68 (2, 12, 14). Moreover, Sam68 poly(U)
binding (11, 45), G8-5 (this study), and REV-like function (7) are
negatively regulated by tyrosine kinases. The absence of secondary
structure in poly(U) and the systematic evolution of ligands by
exponential enrichment RNAs such as G8-5 and the lack of
defined sequence/structure in the RRE recognized by Sam68 suggest that
specific RNA binding, mediated by only the Sam68 GSG domain, may only
be observed in the context of the whole protein when
tyrosine-phosphorylated.
| |
FOOTNOTES |
|---|
* This work was supported in part by Medical Research Council of Canada Grant MT13377.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Supported by a post-doctoral fellowship from the Canadian Institutes of Health Research.
¶ Supported by a fellowship from Consejo Nacional de Ciencia y Technología.
Scholar of the Medical Research Council of Canada. To whom
correspondence should be addressed: Lady Davis Institute, 3755 Côte Ste-Catherine Rd., Montréal, Québec, Canada H3T
1E2. Tel.: 514-340-8260; Fax: 514-340-8295; E-mail:
sricha@po-box.mcgill.ca.
Published, JBC Papers in Press, June 6, 2001, DOI 10.1074/jbc.M102247200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: Sam68, Src associated substrate in mitosis of 68 kDa; KH, heterogeneous nuclear ribonucleoprotein K homology; GLD-1, germline defective-1; GRP33, glycine-rich protein of 33 kDa; GSG, GRP33, Sam68, GLD-1; NK region, N-terminal of KH domain; CK region, C-terminal of KH domain; STAR, signal transduction and activator of RNA metabolism; QKI, quaking; PCR, polymerase chain reaction; UTR, untranslated region; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; RRE, REV response element; CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-galactosidase
expression vector. CAT activity was normalized for
