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A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth.

Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J - J. Cell Biol. (1997)

Bottom Line: 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.When the culture temperature was lowered from 37 to 32 degrees C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37 degrees C.By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37 degrees C with prolongation of G1 phase of the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

ABSTRACT
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 degrees C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37 degrees C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37 degrees 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.

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Localization of  CIRP. Immunofluorescence  microscopy of BALB/3T3  cells cultured at 32°C and  stained with an anti-CIRP  polyclonal antibody (a) or  preimmune serum (c). The  bound antibody was detected  by an FITC-conjugated second antibody. Fluorescence  microscopy of COS-7 cells  expressing GFP–CIRP fusion protein (e) or GFP (g).  Light (b, d, and f) or phasecontrast (h) microscopic images of the field of view identical to a, c, e, and g, respectively. Bars, 20 μm.
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Figure 8: Localization of CIRP. Immunofluorescence microscopy of BALB/3T3 cells cultured at 32°C and stained with an anti-CIRP polyclonal antibody (a) or preimmune serum (c). The bound antibody was detected by an FITC-conjugated second antibody. Fluorescence microscopy of COS-7 cells expressing GFP–CIRP fusion protein (e) or GFP (g). Light (b, d, and f) or phasecontrast (h) microscopic images of the field of view identical to a, c, e, and g, respectively. Bars, 20 μm.

Mentions: Immunofluorescence microscopy using the anti-CIRP polyclonal antibody showed that signals were detected in the nucleoplasm of almost all BALB/3T3 cells cultured at 32°C (Fig. 8, a and b), while no signals were detected using preimmune serum (Fig. 8, c and d). To confirm this result, we fused cirp and Aequorea GFP cDNAs in an expression vector. GFP has been used as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells (Ogawa et al., 1995). When GFP–CIRP expression vector DNA was transfected into COS-7 cells, fluorescence was found only in the nucleoplasm (Fig. 8, e and f). On the other hand, when GFP expression vector DNA was transfected, fluorescence was detected in cytoplasm and nucleus (Fig. 8, g and h). These results suggest that CIRP is localized in the nucleoplasm.


A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth.

Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J - J. Cell Biol. (1997)

Localization of  CIRP. Immunofluorescence  microscopy of BALB/3T3  cells cultured at 32°C and  stained with an anti-CIRP  polyclonal antibody (a) or  preimmune serum (c). The  bound antibody was detected  by an FITC-conjugated second antibody. Fluorescence  microscopy of COS-7 cells  expressing GFP–CIRP fusion protein (e) or GFP (g).  Light (b, d, and f) or phasecontrast (h) microscopic images of the field of view identical to a, c, e, and g, respectively. Bars, 20 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139845&req=5

Figure 8: Localization of CIRP. Immunofluorescence microscopy of BALB/3T3 cells cultured at 32°C and stained with an anti-CIRP polyclonal antibody (a) or preimmune serum (c). The bound antibody was detected by an FITC-conjugated second antibody. Fluorescence microscopy of COS-7 cells expressing GFP–CIRP fusion protein (e) or GFP (g). Light (b, d, and f) or phasecontrast (h) microscopic images of the field of view identical to a, c, e, and g, respectively. Bars, 20 μm.
Mentions: Immunofluorescence microscopy using the anti-CIRP polyclonal antibody showed that signals were detected in the nucleoplasm of almost all BALB/3T3 cells cultured at 32°C (Fig. 8, a and b), while no signals were detected using preimmune serum (Fig. 8, c and d). To confirm this result, we fused cirp and Aequorea GFP cDNAs in an expression vector. GFP has been used as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells (Ogawa et al., 1995). When GFP–CIRP expression vector DNA was transfected into COS-7 cells, fluorescence was found only in the nucleoplasm (Fig. 8, e and f). On the other hand, when GFP expression vector DNA was transfected, fluorescence was detected in cytoplasm and nucleus (Fig. 8, g and h). These results suggest that CIRP is localized in the nucleoplasm.

Bottom Line: 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.When the culture temperature was lowered from 37 to 32 degrees C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37 degrees C.By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37 degrees C with prolongation of G1 phase of the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

ABSTRACT
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 degrees C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37 degrees C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37 degrees 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.

Show MeSH
Related in: MedlinePlus