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Laboratory of Cellular and Molecular Biology,1 Research Resources Branch,2 Laboratory of Genetics, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 212243
Received 15 May 2005/ Returned for modification 8 June 2005/ Accepted 8 August 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs
have not been systematically identified. Here, immunoprecipitation (IP)
of TIA-1-RNA complexes, followed by microarray-based
identification and computational analysis of bound transcripts, was
used to elucidate a common motif present among TIA-1 target mRNAs. The
predicted TIA-1 motif was a U-rich, 30- to 37-nucleotide (nt)-long
bipartite element forming loops of variable size and a bent stem. The
TIA-1 motif was found in the TNF- and COX-2
mRNAs and in 3,019 additional UniGene transcripts (
3% of the
UniGene database), localizing preferentially to the 3'
untranslated region. The interactions between TIA-1 and target
transcripts were validated by IP of endogenous mRNAs, followed by
reverse transcription and PCR-mediated detection, and by pulldown of
biotinylated RNAs, followed by Western blotting. Further studies using
RNA interference revealed that TIA-1 repressed the
translation of bound mRNAs. In summary, we report a signature motif
present in mRNAs that associate with TIA-1 and provide support to the
notion that TIA-1 represses the translation of target
transcripts. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TIA-1 has been
reported to participate in the regulation of alternative pre-mRNA
splicing of bound mRNAs
(18,
19). However, TIA-1 has
been best characterized as a suppressor of translation, as shown for
the target ARE-bearing mRNAs encoding tumor necrosis factor alpha
(TNF-) and cyclooxygenase 2 (COX-2)
(15,
35). Following
stimulation with bacterial lipopolysaccharide, macrophages derived from
either wild-type or TIA-1–/– mice expressed
the same levels of TNF- mRNA, but
TIA-1–/– cells expressed much more
TNF- protein than cells expressing TIA-1. In
TIA-1–/– macrophages, the levels of
TNF- mRNA found in polysomes were significantly
higher, lending further support to the notion that TIA-1 functions as a
translational silencer
(35). Similarly, the
steady-state levels of COX-2 mRNA were the same in
TIA-1-expressing and -deficient fibroblasts, but cells lacking TIA-1
had significantly higher levels of COX-2 mRNA in polysomes and
expressed elevated levels of COX-2 protein
(15).
The
mechanisms whereby TIA-1 represses translation have been investigated
most extensively in cells responding to environmental stress agents.
Stress-triggered translational inhibition is characterized by the
activation of one or more protein kinases (PKR, PERK, GCN2, and HRI)
that phosphorylate the subunit of eukaryotic initiation
factor 2 (eIF-2), a constituent of the ternary complex
(eIF-2-GTP-
)
that loads initiator
onto the small ribosomal subunit to initiate protein translation
(14,
27). Phosphorylated
eIF-2 inhibits translation by reducing the availability of the
active ternary complex; under these conditions, TIA-1 has been proposed
to interact with the translational machinery on the 5' region
of the mRNA and to promote the assembly of noncanonical,
translationally incompetent initiation complexes
(3). In situations of
stress, when many transcripts are simultaneously subject to such
translational silencing, the self-aggregating properties of TIA-1
promote the formation of cytoplasmic foci known as stress granules
(SGs), which are generally believed to represent sites of translational
inhibition (26).
Nonetheless, the underlying translational control mechanisms mediated
by TIA-1 are likely to be similar in stressed and unstressed cells
(1,
2). In the case of mRNAs
bearing 3' untranslated region (3'UTR) AREs that form
complexes with TIA-1 (TNF- and COX-2), the likelihood will be
greater that translationally silent preinitiation complexes assemble on
the 5' region of the transcript, and therefore ARE-bearing
mRNAs would be preferentially subject to translational repression
(3).
Given that TIA-1 is implicated in critical cellular events, including the response to stress agents, apoptotic stimuli, and inflammatory factors, we thought it would be highly valuable to systematically identify the collection of TIA-1 target mRNAs. Here, we describe efforts undertaken to elucidate such TIA-1-associated transcripts in human colorectal cancer cells using en masse methodologies. The analysis was carried out by immunoprecipitating TIA-1-RNA complexes from stressed cells and identifying the bound transcripts using microarrays. Computational analysis of the target transcripts led to the elucidation of a shared, U-rich motif present in TIA-1 target mRNAs. The data revealed that novel TIA-1 target mRNAs can be successfully identified using this motif and that mRNAs associating with TIA-1 are translationally repressed.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IP assays. Immunoprecipitation (IP) of TIA-1-mRNA complexes from RKO cell lysates was used to assess the association of endogenous TIA-1 with endogenous target mRNAs. The IP assay was performed essentially as described previously (32, 37), except 100 million cells were used as starting material and lysate supernatants were precleared for 30 min at 4°C using 15 µg of immunoglobulin G (IgG) (Santa Cruz Biotechnology) and 50 µl of protein A-Sepharose beads (Sigma) that had been previously swollen in NT2 buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2, and 0.05% Nonidet P-40 [NP-40]) supplemented with 5% bovine serum albumin. Beads (100 µl) were incubated (16 h, 4°C) with 30 µg of antibody (either goat IgG [Santa Cruz Biotechnology] or goat anti-TIA-1 [Santa Cruz Biotechnology]) and then for 1 h at 4°C with 3 mg of cell lysate. After extensive washes and digestion of proteins in the IP material (37), the RNA was extracted and used either for hybridization of cDNA arrays (below) or for verification of TIA-1 target transcripts. For the latter analysis, RNA in the IP material was used to perform reverse transcription-PCR (RT-PCR) to detect the presence of specific target mRNAs using gene-specific primer pairs (available upon request). PCR products were visualized after electrophoresis in 1% agarose gels stained with ethidium bromide. To assess the proteins present in the IP material, the above procedure was followed, except proteins were not digested and were instead extracted from the beads using Laemmli buffer and detected by Western blot analysis.
Where indicated, purified recombinant proteins (either glutathione S-transferase [GST] or GST-TIA-1 at a concentration of 500 nM) were incubated with the precleared cell lysates for an additional 30 min at 45°C, before adding beads that had been precoated with anti-GST antibody. All subsequent steps were as described above, including IP, washes, RT, and PCR amplification.
cDNA array analysis.
RNA in the
material obtained after IP reactions using either an anti-TIA-1
antibody or IgG was reverse transcribed in the presence of
[-33P]dCTP (MP Biomedicals), and the radiolabeled
product was used to hybridize cDNA arrays
(http://www.grc.nia.nih.gov/branches/rrb/dna/index/dnapubs.htm#2,MGC arrays containing 9,600 genes), employing previously reported
methodologies (32,
37,
38). All of the data were
analyzed using the Array Pro software (Media Cybernetics, Inc.), then
normalized by Z-score transformation
(8) and used to calculate
differences in signal intensities. Significant values were tested using
a two-tailed Z-test and a P of 
0.01. The
data were calculated from three independent experiments. The complete
cDNA array data are available from the
authors.
Computational analysis. Human UniGene records were first identified from the most strongly enriched TIA-1 targets derived from the array analysis; the top 185 transcripts from which the 3'UTRs were available (available upon request) served as the experimental data set for the identification of the TIA-1 motif. The 185 3'UTR sequences were first scanned with RepeatMasker (www.repeatmasker.org) to remove repetitive sequences. The remaining sequences were divided into 100-base-long subsequences with 50-base overlap between consecutive sequences and were organized into 50 data sets. Common RNA motifs were elucidated from each of the 50 random data sets. The top 10 candidate motifs from each random data set were selected and were used to build the stochastic context-free grammar (SCFG) model. The SCFG model of each candidate motif was used to search against the experimental 3'UTR data set as well as the entire human UniGene 3'UTR data set to obtain the number of hits for each motif. The motif with the highest enrichment in the experimental data set over the entire UniGene data set was considered to be the best TIA-1 candidate motif. The enrichment was examined by Fisher's exact test. The identification of the RNA motif in unaligned sequences was conducted using FOLDALIGN software (21), and the identified motif was modeled by the SCFG algorithm and searched against the transcript data set using the COVE and COVELS software packages (17). The motif logo was constructed using WebLogo (http://weblogo.berkeley.edu/). RNAplot was used to depict the secondary structure of the representative RNA motifs. The computation was performed using the NIH Biowulf computer farm. Both UniGene and Refseq data sets were downloaded from NCBI.
Synthesis of biotinylated transcripts and analysis of TIA-1 bound to biotinylated RNA. For in vitro synthesis of biotinylated transcripts, reverse-transcribed total RNA was used as the template for PCRs using 5' oligonucleotides that contained the T7 RNA polymerase promoter sequence. All oligonucleotide pairs used to synthesize DNA templates for the production of biotinylated transcripts are available upon request. The following genes (with the amplified regions indicated in parentheses) were assayed for biotin pulldown: ACTG1 (1201 to 1904), PFN1 (556 to 733), ACTB (1219 to 1724), APH-1A (864 to 1910), DEK (1312 to 2077), MTA1 (2018 to 2609), GAPDH (981 to 1283), CALM2 (522 to 1071), SNRPF (345 to 445), CDK9 (1229 to 1732), APEX1 (1314 to 1541), and PTMA (546 to 1202). The PCR-amplified products were resolved on agarose gels, and transcripts were purified and used as templates for the synthesis of the corresponding biotinylated RNAs using T7 RNA polymerase and biotin-CTP (39).
Biotin pull-down assays (39) were carried out by incubating 500 nM of either recombinant GST or GST-TIA-1 proteins (prepared in Escherichia coli using an expression vector kindly provided by J. Varcarcel and P. Anderson [18]) with 0.2 µg of biotinylated transcripts for 30 min at 45°C. Complexes were isolated using streptavidin-conjugated Dynabeads (Dynal), and bound proteins in the pull-down material were analyzed by Western blotting using antibodies recognizing GST (below).
Immunofluorescence. RKO cells cultured on dishes containing coverslips were fixed in 4% formaldehyde (15 min) and permeabilized in cold 0.2% Triton X-100 in phosphate-buffered saline (PBS) (15 min). After incubation in blocking buffer (5% horse serum in PBS) for 1 h at 37°C, coverslips were incubated with either goat anti-TIA-1 or goat anti-TIAR (Santa Cruz Biotechnology) in blocking buffer (1 h at 37°C, 1:200 dilution), washed with PBS containing 0.1% Tween 20, and further incubated with Alexa Fluor 568-labeled donkey anti-goat IgG (heavy plus light chains) (Molecular Probes; 1 h at 37°C, 1:500 dilution). After washes with PBS containing 0.1% Tween 20, coverslips were mounted in Vectashield (Vector Laboratories) and visualized with a Zeiss LSM410 confocal microscope. Representative photographs from three independent experiments are shown. Negative-control incubations were performed without primary antibody.
Analysis of newly translated protein. Newly translated CALM2, SNRPF, CASP8, and (control) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed by incubating 4 x 106 cells with 1.5 mCi L-[35S]methionine and L-[35S]cysteine (Easy Tag EXPRESS; NEN/Perkin Elmer) per 100-mm plate for 20 min, whereupon cells were lysed using TSD lysis buffer (50 mM Tris [pH 7.5], 1% sodium dodecyl sulfate [SDS], and 5 mM dithiothreitol). IP reactions were carried out as previously described (33) for 1 h at 4°C using appropriate antibodies and IgG as a control. Following extensive washes in TNN buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 0.5% NP-40, 250 mM NaCl), the IP material was resolved by either 15% (for CALM2 and SNRPF) or 10% (for CASP8) SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride filters, and visualized using a PhosphorImager (Molecular Dynamics).
Western blot analysis.
The preparation
of whole-cell, cytoplasmic, and nuclear lysates was previously
described (28,
40). Protein lysates (5
to 20 µg) were resolved by SDS-polyacrylamide gel
electrophoresis and transferred onto nitrocellulose membranes.
Antibodies were used to detect -tubulin, hnRNP C1/C2, S6, GST,
TIAR, or TIA-1 (Santa Cruz Biotechnology), CASP8 (BD Pharmingen), CALM2
(Upstate Cell Signaling Solutions), or SNRPF (a gift from D.
Ingelfinger). Following secondary antibody incubations, signals were
visualized by enhanced
chemiluminescence.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3% of the total spots on the array) had Z scores of
>1.00 in a comparison of the signals in TIA-1 IP arrays with
those in IgG IP arrays and were thus deemed to represent specific
TIA-1-associated transcripts. Of the specific TIA-1-associated
transcripts, the 185 transcripts for which full-length mRNAs were
available (the experimental data set [available upon request]) were
selected for further analysis.
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23% of
the experimental data set) had hits for the TIA-1 motif. The fact that
the remaining 143 putative TIA-1 target transcripts from the
experimental data set did not appear to contain the 30- to 37-base
motif suggested that additional TIA-1 motifs may also exist that were
not identified in this analysis. In this regard, it should also be
explained that other motifs which had a greater number of hits in the
experimental data set were identified, but the hit frequency was lower
relative to the hit frequency within the UniGene database and were thus
considered to be less strong TIA-1 motifs. Importantly, the two mRNAs
that have been shown to be targets of TIA-1 (those encoding
TNF- and COX-2
[10,
35]) were found to
contain at least one motif hit (Table
2).
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To ascertain
whether the presence of the TIA-1 motif was sufficient for TIA-1 to
associate with a given RNA, we synthesized two sets of biotinylated
transcripts (Fig. 4A,
schematic). The first were three small RNAs comprising either the TIA-1
motif from the TNF- mRNA (similar results were
observed with the TIA-1 motif present in the CASP8 mRNA [data not
shown]) plus 20-base flanking RNA from each side of the motif
[TIA-1(+)], only the flanking regions [TIA-1(–)], or a
mutated TIA-1 motif with flanking regions [TIA-1(mut)]. In the second
set, we tested whether the presence of the TIA-1 motif would render a
heterologous, nontarget transcript (the GAPDH 3'UTR) capable of
forming complexes with TIA-1. As shown in Fig.
4B, only transcripts
bearing the TIA-1(+) motif in each transcript set showed
specific interaction with GST-TIA-1. No binding was seen when
the TIA-1 motif was absent [TIA-1(–) transcripts] or when it
was mutated [TIA-1(mut) transcripts], indicating that the presence of
an intact TIA-1 motif was required for binding. No complexes were
detected in control incubations with GST.
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TIA-1 knockdown relieves the translation of target mRNAs. Additional insight into the association of TIA-1 with target mRNAs and the functional consequences of these interactions was sought by RNA interference (RNAi)-mediated reduction of TIA-1 levels. The effects of four different small interfering RNAs targeting TIA-1 are shown (Fig. 5A; T1 was used in subsequent experiments). Transfection with each of the siRNA molecules caused a dramatic reduction in TIA-1 levels (to less than 5% of the levels seen in control siRNA populations). It was important to assess the levels of TIAR in the TIA-1 siRNA populations, given the extensive sequence homology between the two proteins; as shown, TIAR levels remained unaltered (Fig. 5A). In keeping with this reduction in TIA-1 levels, RT-PCR amplification of putative target transcripts encoding CALM2, SNRPF, and CASP8 was markedly reduced when using IP material obtained from the TIA-1 siRNA population (Fig. 5B). Analysis of TIA-1 and TIAR by immunofluorescence confirmed the effects of RNAi on TIA-1 expression and further showed that SGs were readily detectable in the TIA-1 siRNA population (as seen by TIAR fluorescence), suggesting that TIAR alone may be sufficient for the assembly of SGs (Fig. 6).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and COX-2 mRNAs, two reported TIA-1
target transcripts. To assess the usefulness of the TIA-1 motif for identifying bona fide target mRNAs, several validation approaches were undertaken. First, the mRNAs present in the TIA-1-associated IP material were tested by RT-PCR analysis using sequence-specific primers; among the particular target mRNAs examined were several transcripts that were identified during the initial cDNA array analysis and several that were predicted to be targets after the UniGene database was searched for transcripts containing the TIA-1 motif. By this methodology, all of the predicted targets were validated (Fig. 3A), except for two array transcripts which did not display appreciable enrichment in the TIA-1 IP material (not shown). In the second set of validation efforts, we sought to determine whether putative TIA-1 target transcripts, synthesized in the presence of biotinylated CTP, formed complexes with recombinant TIA-1. All of the predicted TIA-1-mRNA interactions were also validated by this in vitro assay (Fig. 3B); only biotinylated SLAMF1 3'UTR transcript did not show the expected binding to TIA-1, despite being enriched by IP plus RT-PCR analysis (not shown), suggesting that perhaps the recombinant transcript did not retain the proper native conformation of the SLAMF1 mRNA. Importantly, when the TIA-1 motif was artificially linked to a heterologous RNA (the nontarget GAPDH 3'UTR), the resulting chimeric transcript was rendered a TIA-1 target; binding was undetectable or at background levels when the motif was absent or mutated (Fig. 4). These experiments further support the validity of the TIA-1 motif elucidated in this study. An additional line of investigation used to substantiate the existence of these interactions assessed the subcellular colocalization of TIA-1 and target SNRPF transcript. The in situ hybridization signals for SNRPF RNA and the immunofluorescent TIA-1 signals (available upon request) overlapped at the sites of SGs. Taken together, the TIA-1 motif reported here successfully identified 23% of TIA-1 target RNAs.
TIA-1 has been reported to participate in the regulation of bound transcripts in at least two distinct processes: pre-mRNA splicing and translational suppression. TIA-1 was previously shown to regulate the splicing of several mRNAs, including the TIA-1 pre-mRNA itself and those encoding fibroblast growth factor receptor 2 and the Fas receptor (11, 18, 29). In this regard, it should be noted that no TIA-1 motif hits were found on either the TIA-1 mRNA or the fibroblast growth factor receptor 2 mRNA (data not shown), although TIA-1 motifs were found on two Fas receptor isoforms (TNFRSF6 and ARTS-1). In this investigation, we have not directly tested whether the mRNAs bearing the motif described here are also targets of TIA-1-regulated splicing. Since the array-based identification of TIA-1 target transcripts was conducted selectively on poly(A)-containing RNA (Materials and Methods), we anticipate that only mature mRNA species were detected on the cDNA arrays. Accordingly, it remains to be directly tested whether a different motif signals the TIA-1-dependent regulation of pre-mRNA splicing.
However, TIA-1 has been
characterized most extensively as a translational suppressor. The
findings reported here indeed support this role for TIA-1, as the
translation of the target mRNAs studied (CALM2, SNRPF, and CASP8) was
enhanced when TIA-1 levels were knocked down by RNA interference (Fig.
7B and C). The
TIA-1-mediated translational suppression likely relies on the ability
of TIA-1 to promote the formation of noncanonical preinitiation
complexes by usurping the position of the ternary complex
(eIF-2-GTP-)
at the 5'UTR of an mRNA. Whereas the active ternary complex
(featuring unphosphorylated eIF-2) promotes the initiation of
translation, TIA-1 instead triggers the aggregation of TIA-1-associated
ribonucleoprotein complexes into translationally silent SGs
(2). In order to assess
the influence of TIA-1 on the translation of target mRNAs, we have
employed a methodology for assessing nascent protein biosynthesis which
measures the pulse incorporation of 35S-labeled amino acids
onto nascent polypeptide chains. This method uniquely provides a
measure of new translation but has major limitations, as it can be used
only to analyze abundant proteins for which highly sensitive and
specific antibodies are available. Approximately one dozen additional
antibodies were tested to assess as many additional proteins encoded by
putative TIA-1 target mRNAs. Unfortunately, the IP signals in each case
were well below the levels of detection of the assay (not shown), and
therefore, the nascent translation of the corresponding proteins could
not be studied by this approach. A comprehensive assessment of
the translation of TIA-1's target mRNAs thus awaits the development of
more sensitive methods.
The changes in abundance of
the CASP8, CALM2, and SNRPF proteins in cells expressing different
levels of TIA-1 determined by Western blotting (Fig.
7C) mirrored those
observed when measuring nascent translation (Fig.
7B), suggesting that the
changes in protein abundance are indeed linked to the changes in
protein biosynthesis influenced by TIA-1. Interestingly, our findings
support the notion that TIA-1 functions as a translational inhibitor
even in the absence of stress, since the nascent translation of CASP8,
CALM2, and SNRPF was elevated in TIA-1 knockdown cells that had been
left without HS. These results are also in agreement with earlier
observations that in unstimulated macrophages derived from
TIA-1–/– mice, the levels of
TNF- mRNA found in polysomes were higher than those
seen in macrophages from wild-type mice, and the
TIA–/– cells expressed elevated levels of
the cytokine (35). In our
studies, HS did not seem to increase the levels of TIA-1 in the
cytoplasm (Fig. 1B) or
promote the binding of TIA-1 to target mRNAs (data not shown). Thus,
the mechanism(s) whereby HS silences target mRNA translation in a
TIA-1-dependent fashion, including possible TIA-1 posttranslational
modification through phosphorylation or its association with other
proteins, remains to be formally investigated. After the 1-hour HS
treatment, however, it is unlikely that the pronounced decline in the
levels of these three proteins is due solely to reduced translation
rates (Fig. 7B); instead,
it is likely to be influenced by altered proteolysis or other
posttranslational events. In light of these observations, we propose
that TIA-1 contributes to altering protein expression by influencing
the biosynthesis of encoded proteins. This level of regulation likely
functions in juxtaposition with additional processes controlling
protein levels, such as subcellular protein transport,
proteolysis, and/or protein secretion.
It is noteworthy
that in populations in which TIA-1 was knocked down (Fig.
5), SGs were
still detected (Fig. 6);
similarly, silencing of TIAR failed to block SG formation (K.
Mazan-Mamczarz and M. Gorospe, unpublished data). Whereas
TIA-1 and TIAR appear to have interchangeable roles regarding SG
formation, their relative affinities for target mRNAs have not been
compared systematically. En masse efforts to identify TIAR and TIA-1
target mRNAs under way in our laboratory have indicated the existence
of both shared and specific target ARE-containing transcripts (I.
López de Silanes, K. Mazan-Mamczarz, and M. Gorospe, unpublished
data), in keeping with the notion that TIAR and TIA-1 are functionally
distinct (42), although
they both appear to bind to several common targets, such as
the COX-2 and TNF- mRNAs.
In summary, we have systematically identified many TIA-1 target mRNAs and describe a common RNA motif among them. Using a variety of approaches, the association of TIA-1 with mRNAs that were either detected as microarray targets or identified on the basis of the presence of the TIA-1 motif in the UniGene database transcripts was validated using several approaches. Importantly, TIA-1 was found to repress the translation of target mRNAs. These discoveries provide comprehensive and valuable insight into the ribonucleoprotein complexes that govern gene expression at the posttranscriptional level.
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ACKNOWLEDGMENTS |
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This research was supported by the Intramural Research Program of the NIA, NIH.
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FOOTNOTES |
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