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© The Rockefeller University Press,
0021-9525/1997//899 $5.00
The Journal of Cell Biology, Volume 137, Number 4,
, 1997 899-908
Article |
A Glycine-rich RNA-binding Protein Mediating Cold-inducible Suppression of Mammalian Cell Growth
Department of Urology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32°C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37°C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37°C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells.
Abbreviations used in this paper: CIRP, cold-inducible RNA-binding protein; CS-RBD, consensus sequence RNA-binding domain; GFP, green fluorescence protein; GRP, glycine-rich RNA-binding protein; GST, glutathione-S-transferase; ODN, oligodeoxynucleotide; RNP, ribonucleoprotein.
To adapt themselves to environmental temperature shifts, organisms have developed sophisticated strategies. To survive in the winter, some animals have developed a capacity for adaptive hypothermia (hibernation), in which they lower their body temperatures to a few degrees above the ambient (Willis, 1987; Aloia and Raison, 1989). Although nonhibernating mammals maintain high uniform body temperatures and have a poor tolerance for hypothermia, their cells may have a capacity for a far wider cold tolerance (Michl et al., 1966; Holecková et al., 1968). In mammals as well as other organisms, cold stress changes the lipid composition of cellular membranes and suppresses the rate of protein synthesis and cell growth (Rao and Engelberg, 1965; Sisken et al., 1965; Watanabe and Okada, 1967; Nelson et al., 1971; Nishimune and Komatsu, 1972; Burdon, 1987; Aloia and Raison, 1989). In clinics, hypothermia is now employed in heart and brain surgeries and in the preservation of donor organs (Fuller, 1987). However, the molecular mechanisms regulating the response to cold stress in mammalian cells are just beginning to be understood (Glofcheski et al., 1993; Matz et al., 1995).
The cold-shock responses of microorganisms have been extensively investigated (Jones et al., 1987; Goldstein et al., 1990; Jones and Inouye, 1994). Cold stress induces the synthesis of several cold-shock proteins, which are involved in various cellular processes such as transcription, translation, and DNA recombination (Jones and Inouye, 1994). Recently, a family of proteins consisting of one amino-terminal consensus sequence RNA-binding domain (CSRBD)1 and one carboxyl-terminal glycine-rich domain has been isolated from cyanobacterium (Sato, 1994, 1995), plant cells (Gómez et al., 1988; Bergeron et al., 1993; Hirose et al., 1993; Carpenter et al., 1994; Heintzen et al., 1994), and human (Derry et al., 1995). This protein family is referred to as a glycine-rich RNA-binding protein (GRP) family and some of them have been demonstrated to be induced by cold stress (Bergeron et al., 1993; Carpenter et al., 1994; Heintzen et al., 1994).
CS-RBD, also referred to as ribonucleoprotein (RNP) motif, RNP consensus sequence, or RNA recognition motif, is one of the major RNA-binding motifs and is the most widely found and best characterized (Kenan et al., 1991; Burd and Dreyfuss, 1994). It is composed of 
90 amino acids, including two highly conserved sequences, an octamer designated RNP1 and a hexamer designated RNP2, and a number of other, mostly hydrophobic conserved amino acids interspersed throughout the motif. A number of proteins with CS-RBD have been found in organisms ranging from Escherichia coli to human (Fukami-Kobayashi et al., 1993). They contain one to four CS-RBDs and auxiliary domains that are characterized by an abundance of specific residues in their amino acid sequences (Bandziulis et al., 1989). Proteins with CS-RBD are involved in the posttranscriptional regulation of gene expression (Burd and Dreyfuss, 1994). For example, poly(A)-binding proteins have one CS-RBD and a proline-rich domain and regulate mRNA stability and translation. SF2/ASF, B52, X16, and PR264/ SC35, whose CS-RBDs make a cluster in the phylogenetic tree, have a serine-arginine–rich domain and are splicing factors. Some hnRNPs, such as hnRNP A1, have two CSRBDs and a glycine-rich domain and play important roles in mRNA biogenesis, such as splicing and RNA export. Although GRPs, hnRNPs, and nucleolin have domains rich in glycine in common, they belong to phylogenetically distinct families, and no definite roles played by GRP have been clarified (Bergeron et al., 1993; Fukami-Kobayashi et al., 1993; Carpenter et al., 1994; Heintzen et al., 1994).
In the present study, we have screened for RNA-binding proteins expressed in mouse testis with a PCR-based cloning method and isolated a cDNA encoding a novel GRP. This protein was induced in response to cold stress and designated as cold-inducible RNA-binding protein (CIRP). Furthermore, we demonstrated that CIRP is involved in temperature-dependent regulation of cell growth.
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Materials and Methods |
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For temperature-shift experiments, BALB/3T3 cells (5 x 103 cells/cm2) were grown for 24 h at 37°C and then shifted to 15, 25, 32, 37, 39, or 42°C in a humidified atmosphere of 5% CO2 in air. After 6 h or other indicated times, the cells were scraped and frozen in liquid nitrogen. Extraction of total RNA, Northern blot analysis, and Western blot analysis were carried out as described (Furutani et al., 1995).
To assess the effects of antisense oligodeoxynucleotide (ODN), sense ODN (0.5 µM), antisense ODN (0.5 µM), or vehicle alone was added to the BALB/3T3 cells in exponential phase of growth, and the temperature was shifted from 37 to 32°C. After 12 h of incubation, the expression of p18cirp was analyzed by Western blotting. To assess the effects on growth, 5 x 103 BALB/3T3 cells were plated into each well of a 24-well plate. 16 h later, 0.1, 0.5, or 1.0 µM of antisense ODN, sense ODN, or vehicle alone was added to each well and the culture was continued. 2 d later, the numbers of cells were determined under a microscope. The experiments were repeated three times, and each assay was performed in triplicate. Statistical differences between sample means were calculated by analysis of variance, followed by unpaired Student's t test. The results are expressed as the mean ± SEM, and P < 0.02 was considered significant.
ODNs
Antisense and sense ODNs containing phosphorothioates were purchased from Cruachem (Kyoto, Japan). The sequence of the sense ODN was (5'-AGCGGCTGCCATGGCATCAG-3') spanning the translation initiation site of cirp mRNA, and that of antisense ODN was complementary to this sequence.
Cloning of cirp cDNA
The first-strand cDNA was prepared from the total cellular RNA of mouse testis (4-mo-old) by using a First-strand cDNA Synthesis Kit (Pharmacia LKB Biotechnology, Piscataway, NJ) with random primers, according to the manufacturer's instructions. For the PCR, we used a set of degenerate primers, (5'-TCGAATTCIAT[C]A[G]AAIC[G]CA[G]AAICC-3' [I; inosine]) encoding the amino acid sequence GFG/AFV/I and (5'TCAAGCTTTTC[T]A[G]TIGGIGGIC[T]T-3') encoding the amino acid sequence F/YV/IGGL. The PCR reaction was repeated for 40 cycles at 94, 37, and 72°C for periods of 1 min. The PCR products were cloned into the EcoRV site of TA vector and then sequenced. Using the cloned PCR fragment as a probe, clone RNP78 was isolated from a mouse testis cDNA library constructed in 
-Zap phage vectors (Stratagene, La Jolla, CA). The nucleotide sequence of RNP78 insert (cirp) was compared to known sequences in GenBank using the Genetics Computer Group software package.
RNA Extraction and Northern Blot Hybridization
Cells were dissolved in TRIzol reagent (Life Technologies, Grand Island, NY), and RNA was extracted by following the manufacturer's instructions. 20 µg of total RNA of each sample was separated in 1.0% agarose/ formaldehyde gels by electrophoresis and was blotted onto nylon filters (Hybond-N+; Amersham International, Buckinghamshire, UK). The filters were hybridized with [
-32P]dCTP-labeled random-primed cDNA fragments, then washed under stringent conditions (65°C for 30 min in a washing buffer composed of 0.1 x SSC and 0.1% SDS), and detected by autoradiography. A 650-bp SacI-EcoRI fragment of cirp cDNA was used as a probe. The filters were stripped and rehybridized with a cDNA probe for the S26 ribosomal protein as an internal control.
Southern Blot Analysis
ZOO-BLOT was purchased from Clontech Laboratories, Inc. (Palo Alto, CA). The ZOO-BLOT contained EcoRI-digested genomic DNAs from nine eukaryotic species: human, monkey, rat, mouse (BALB/c), dog, cow, rabbit, chicken, and yeast (Saccharomyces cerevisiae). For Southern blot analysis of mouse genomic DNA, DNA was extracted from the mouse liver of 4-mo-old ddy/std and C57BL/6 mice as previously described (Kaneko et al., 1995). The extracted DNA was thoroughly digested with one of the restriction enzymes, EcoRI, PvuII, or KpnI. 20 µg of the digested DNA was electrophoresed in 0.8% agarose gel and transferred onto a nylon filter (Hybond-N+; Amersham International). Filters were hybridized with an [-32P]dCTP-labeled probe at 65°C and washed under high-stringent conditions (two times in 0.1 x SSC and 0.1% SDS at 65°C). The coding region of cirp cDNA, positions 82–600, was used as a probe.
Protein Analysis
The anti-CIRP polyclonal antibody was produced by immunizing rabbits with a carboxyl-terminal oligopeptide (DSYDSYATHNE). For Western blot analysis, 10 µg of proteins were separated by 14% SDS-PAGE, blotted onto polyvinyl difluoride membranes (Millipore, Tokyo, Japan), and treated with the antiserum (1:20,000 diluted). Bound antibody was detected using a goat anti–rabbit IgG conjugated with horseradish peroxidase (BioRad Labs, Hercules, CA) and the enhanced chemiluminescence system (Amersham International). Protein concentrations were determined using the Bradford reagent (BioRad Labs).
Fluorescence Microscopy
Immunofluorescence microscopy was performed on BALB/3T3 cells as previously described by Osborn (1994). The anti-CIRP polyclonal antibody was used at a dilution of 1:10,000. Detection was with FITC-conjugated goat anti–rabbit IgG (DAKO) diluted 1:1,000. As a control, preimmune serum was used at the same dilution.
Green fluorescence protein (GFP)/Thr65 was purchased from Clontech Laboratories Inc., and was ligated into MluI and NotI sites of pMkit-neo vector (pGFP). Cirp cDNA encoding amino acids 1–162 was ligated inframe into PstI site of GFP/Thr65 (pGFP–CIRP). To assess the intracellular localization of GFP–CIRP fusion protein and GFP, we transfected pGFP–CIRP or pGFP DNA into COS-7 cells. After 3 d of culture at 37°C, the medium was removed and replaced with PBS. Then the cells were examined by using a fluorescence microscope (Olympus, Tokyo, Japan) equipped with the FITC filter set.
Northwestern Analysis
A glutathione-S-transferase (GST)–fusion protein was prepared by cloning a 519-bp PCR fragment (positions 83–601) of cirp cDNA into the SmaI site of the expression vector pGEX-4T1 (Pharmacia LKB Biotechnology). Transformed DH5 E. coli cells were grown at 30°C and GST– CIRP fusion protein was purified with Bulk GST Purification Modules (Pharmacia LKB Biotechnology) following the manufacturer's instructions. 2 µg of GST or 1 µg of GST-fusion protein per lane was electrophoresed on 12% SDS-PAGE gels, one of which was stained with Coomassie blue. Others were blotted onto PVDF membrane (Millipore) and denatured and renatured with guanidine-HCl methods, and Northwestern analysis was carried out as described by Schumacher et al. (1995). Each RNA homopolymer probe (Pharmacia LKB Biotechnology) was labeled with [
-32P]ATP and incubated with the blot. To examine the strength of RNA binding, the blots were subsequently washed in the binding buffer containing increasing concentrations of NaCl up to 2.0 M.
Flow Cytometry
To analyze the effect of CIRP on cell cycle, we cloned the cirp cDNA into the expression vector pHβApr-1 (Gunning et al., 1987) in the forward orientation relative to the β-actin promoter (pHβApr-1-CIRP). BALB/3T3 cells were transfected with the pHβApr-1-CIRP DNA or the pHβApr-1 DNA. Stable clones were selected in G418 and by limiting dilutions. Five p18cirp-overexpressors and seven vector-transfected controls were analyzed. Doubling times were determined from the growth curves. For cell cycle analysis, trypsinized cell suspensions were adjusted to a concentration of 1 x 106 cells/ml in prechilled 70% ethanol. Samples stored at 4°C overnight were stained with propidium iodide and analyzed by Epics Elite flow cytometer (Coulter Corp., Hialeah, FL). A minimum of 10,000 events were collected, and cell cycle distribution was obtained using a computer modeling program (Multicycle AV; Phoenix Flow Systems, San Diego, CA). Experiments were repeated three times. Length of G1 phase was calculated from the percent of cells in G1 phase according to the equation Td x {1 – log [2 – F(G1)]/log 2}, in which Td is the doubling time and F(G1) is the fraction of cells in G1 phase (Koyasu et al., 1989).
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1.3 and 2.9 kb in size derived from mouse testis (data not shown). RNP78 (cirp) contained a 1,264-bp insert with an open reading frame potentially encoding 172 amino acids (Fig. 1). The sequence around the first ATG codon at nucleotide position 81 provided a favorable context for translation initiation (Kozak, 1991), and the 5' untranslated region contained an in-frame stop codon at position 10. The predicted amino acid sequence displayed two main features: the presence of an amino-terminal CS-RBD and a carboxyl-terminal glycine-rich domain. The CS-RBD of cirp contained consensus sequences of RNP1, RNP2, and a number of other, mostly hydrophobic conserved amino acids interspersed throughout the motif (Fig. 2 a). The carboxyl-terminal part was rich in glycine, serine, arginine, and tyrosine (38.8, 16.4, 19.4, and 10.4%, respectively).
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To assess the evolutionary conservation of the cirp gene, Southern blot analysis of genomic DNAs from nine eukaryotic species, human, monkey, rat, mouse, dog, cow, rabbit, chicken, and yeast, was carried out using the coding region of cirp cDNA as a probe. As shown in Fig. 3 a, signals were detected in all samples under high-stringent conditions, suggesting that the cirp gene family is well conserved beyond species. When the genomic DNA samples from two different strains of mice were digested with one of three restriction enzymes—EcoRI, PvuII, or KpnI— and analyzed, a single band was detected in each DNA sample (Fig. 3 b). Thus, the cirp gene is probably a singlecopy gene in mice.
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Discussion |
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CIRP consists of CS-RBD and a glycine-rich domain. Binding experiments with ribohomopolymers and various RNAs have established that most of the CS-RBD–containing proteins have distinct RNA-binding characteristics (Burd and Dreyfuss, 1994). The Northwestern assay indicated that CIRP preferentially bound to poly(U). Similar nucleic acid–binding properties are known for hnRNP C, hnRNP A1, RGP-1b, and RGP-2 (Swanson and Dreyfuss, 1988a; Hirose et al., 1993, 1994). CIRP was localized to the nucleoplasm as known for hnRNP C and hnRNP A1 (Dreyfuss et al., 1984; Pin
l et al., 1989). The hnRNP C binds preferentially to U-rich polypyrimidine tracts found at the 3'-ends of introns and in 3' untranslated regions of mRNAs and is suggested to be important for 3'-end cleavage and polyadenylation (Swanson and Dreyfuss, 1988b; Wilusz et al., 1988). The hnRNP A1 is suggested to be involved in the 5'-splice site selection in an alternative splicing and mRNA transport (Mayeda and Krainer, 1992; Pinl and Dreyfuss, 1992). In a similar fashion, CIRP may control the specific or general gene expression independently or by competing with hnRNP C and/or hnRNP A1 for the target binding sequences.
Recent studies on cold-shock response of E. coli have shown that CsdA, one of the major cold-shock proteins associated with ribosome, has a helix-destabilizing activity, and the csdA deletion impairs cell growth and the synthesis of a number of proteins at low temperature (Jones et al., 1996). The secondary structures in mRNAs that inhibit translation initiation are considered to become more stable at low temperature and are deleterious for growth of E. coli (Jones and Inouye, 1994). By unwinding stable secondary structures in mRNAs as an RNA chaperone and thus facilitating ribosomal functions, CsdA is supposed to play an essential role in mRNA translation at low temperature (Jones et al., 1996). The secondary structures and/or RNA–RNA annealing of pre-mRNA/mRNA are also proposed to play a critical role in processing of pre-mRNA into mRNA (Steitz, 1992, 1993; Lamm and Lamond, 1993). Therefore, the cold-inducible helix-destabilizing proteins are probably involved in processing of pre-mRNA at low temperature as well. Interestingly, hnRNP A1, hnRNP C, and its CS-RBD possess the ability to destabilize the helix and/or promote annealing of complementary nucleic acids (Görlach et al., 1992; Portman and Dreyfuss, 1994). It remains to be determined whether CIRP has such an RNA chaperone activity and how reduction of CIRP levels, contrary to the case with CsdA in E. coli, reverse growth inhibitory effects of low temperature in mammalian cells.
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Acknowledgments |
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Submitted: 15 October 1996
Revised: 27 February 1997
This work was partly supported by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan.
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