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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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Temperature-dependent expression of cirp  mRNA.  Northern blot analysis of total RNAs from  mouse cell lines, BALB/3T3  (a) and BMA1 and TAMA26  (b), harvested 24 h after the  indicated temperature shift.  The positions of 18S and 28S  ribosomal RNAs are indicated on the left. As a control  for the amount of RNA  loaded, the filter was rehybridized with a mouse S26 ribosomal protein cDNA probe  (lower).
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Figure 5: Temperature-dependent expression of cirp mRNA. Northern blot analysis of total RNAs from mouse cell lines, BALB/3T3 (a) and BMA1 and TAMA26 (b), harvested 24 h after the indicated temperature shift. The positions of 18S and 28S ribosomal RNAs are indicated on the left. As a control for the amount of RNA loaded, the filter was rehybridized with a mouse S26 ribosomal protein cDNA probe (lower).

Mentions: To investigate the effect of temperature shifts on cirp expression, we exposed BALB/3T3 mouse fibroblasts to heat or cold treatment. As shown in Fig. 5 a, Northern blot analysis indicated that the levels of cirp mRNA increased markedly in response to mild cold treatment (32–25°C) but not to severe cold treatment (15°C). In contrast, heat treatment (39 and 42°C) decreased the levels of expression. Similar results were obtained in two other mouse cell lines, BMA1 bone marrow stromal cells (Fujita et al., 1983) and TAMA26 Sertoli cells (Kaneko et al., 1997) (Fig. 5 b and data not shown). To examine the expression at the protein level, we raised a polyclonal antibody against the predicted carboxyl-terminal peptide of CIRP. The antibody specifically recognized an 18-kD protein as expected from the amino acid sequence in the lysates of BALB/3T3 cells (Fig. 6). In agreement with the results of Northern blot analysis, the protein level was found to be increased 24 h after a temperature shift from 37 to 32°C. Subsequent Western blot analysis demonstrated that the level of p18cirp increased within 6 h after the temperature shift and peaked at 12 h, and the elevated level persisted for more than 24 h (Fig. 7).


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)

Temperature-dependent expression of cirp  mRNA.  Northern blot analysis of total RNAs from  mouse cell lines, BALB/3T3  (a) and BMA1 and TAMA26  (b), harvested 24 h after the  indicated temperature shift.  The positions of 18S and 28S  ribosomal RNAs are indicated on the left. As a control  for the amount of RNA  loaded, the filter was rehybridized with a mouse S26 ribosomal protein cDNA probe  (lower).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Temperature-dependent expression of cirp mRNA. Northern blot analysis of total RNAs from mouse cell lines, BALB/3T3 (a) and BMA1 and TAMA26 (b), harvested 24 h after the indicated temperature shift. The positions of 18S and 28S ribosomal RNAs are indicated on the left. As a control for the amount of RNA loaded, the filter was rehybridized with a mouse S26 ribosomal protein cDNA probe (lower).
Mentions: To investigate the effect of temperature shifts on cirp expression, we exposed BALB/3T3 mouse fibroblasts to heat or cold treatment. As shown in Fig. 5 a, Northern blot analysis indicated that the levels of cirp mRNA increased markedly in response to mild cold treatment (32–25°C) but not to severe cold treatment (15°C). In contrast, heat treatment (39 and 42°C) decreased the levels of expression. Similar results were obtained in two other mouse cell lines, BMA1 bone marrow stromal cells (Fujita et al., 1983) and TAMA26 Sertoli cells (Kaneko et al., 1997) (Fig. 5 b and data not shown). To examine the expression at the protein level, we raised a polyclonal antibody against the predicted carboxyl-terminal peptide of CIRP. The antibody specifically recognized an 18-kD protein as expected from the amino acid sequence in the lysates of BALB/3T3 cells (Fig. 6). In agreement with the results of Northern blot analysis, the protein level was found to be increased 24 h after a temperature shift from 37 to 32°C. Subsequent Western blot analysis demonstrated that the level of p18cirp increased within 6 h after the temperature shift and peaked at 12 h, and the elevated level persisted for more than 24 h (Fig. 7).

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