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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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Expression of p18cirp in representative transfectants.  BALB/3T3 cells, cloned BALB/3T3 cells transfected with cirpexpression vector DNA (BAC-Sn3), or vector DNA (BAC-C1)  were cultured for 12 h at the indicated temperature and analyzed  by Western blotting.
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Figure 11: Expression of p18cirp in representative transfectants. BALB/3T3 cells, cloned BALB/3T3 cells transfected with cirpexpression vector DNA (BAC-Sn3), or vector DNA (BAC-C1) were cultured for 12 h at the indicated temperature and analyzed by Western blotting.

Mentions: To further elucidate the role that p18cirp plays in the regulation of cell growth, a full-length cirp cDNA in an expression vector was stably transfected into BALB/3T3 cells. Five clones overexpressing p18cirp and seven control clones transfected with the vector DNA alone were analyzed. Expressions of p18cirp in a representative clone BAC-Sn3 and a control clone BAC-C1 are illustrated in Fig. 11. The p18cirp level in BAC-Sn3 cells maintained at 37°C was almost equal to that induced in BALB/3T3 cells upon cold stress. When these p18cirp-overexpressing clones and control clones were cultured at 37°C, the doubling time of the former was significantly longer than that of the latter (23.5 vs. 17.6 h, Table I). The cell cycle analysis revealed that the percentage of cells in G1 phase was 45.4% for control transfectants and 53.6% for CIRP transfectants. The percentage of cells in S phase was 40.5% for control transfectants and 32.4% for CIRP transfectants. Therefore, the duration of G1 phase was 6.6 h for control transfectants and 10.5 h for CIRP transfectants, suggesting that the lower growth rate of CIRP transfectants was due to the prolongation of G1 phase. Similar prolongation of G1 phase was observed when BALB/3T3 cells were transferred from 37 to 32°C (6.1 vs. 12.7 h). In addition to prolongation of G1 phase, the duration of S + G2 + M phase was prolonged in BALB/3T3 cells cultured at 32°C, but not in CIRP transfectants cultured at 37°C (Table I). These results indicate that CIRP plays a role in regulation of growth in response to temperature downshifts, but other factors also contribute to the cold-induced growth impairment.


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)

Expression of p18cirp in representative transfectants.  BALB/3T3 cells, cloned BALB/3T3 cells transfected with cirpexpression vector DNA (BAC-Sn3), or vector DNA (BAC-C1)  were cultured for 12 h at the indicated temperature and analyzed  by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: Expression of p18cirp in representative transfectants. BALB/3T3 cells, cloned BALB/3T3 cells transfected with cirpexpression vector DNA (BAC-Sn3), or vector DNA (BAC-C1) were cultured for 12 h at the indicated temperature and analyzed by Western blotting.
Mentions: To further elucidate the role that p18cirp plays in the regulation of cell growth, a full-length cirp cDNA in an expression vector was stably transfected into BALB/3T3 cells. Five clones overexpressing p18cirp and seven control clones transfected with the vector DNA alone were analyzed. Expressions of p18cirp in a representative clone BAC-Sn3 and a control clone BAC-C1 are illustrated in Fig. 11. The p18cirp level in BAC-Sn3 cells maintained at 37°C was almost equal to that induced in BALB/3T3 cells upon cold stress. When these p18cirp-overexpressing clones and control clones were cultured at 37°C, the doubling time of the former was significantly longer than that of the latter (23.5 vs. 17.6 h, Table I). The cell cycle analysis revealed that the percentage of cells in G1 phase was 45.4% for control transfectants and 53.6% for CIRP transfectants. The percentage of cells in S phase was 40.5% for control transfectants and 32.4% for CIRP transfectants. Therefore, the duration of G1 phase was 6.6 h for control transfectants and 10.5 h for CIRP transfectants, suggesting that the lower growth rate of CIRP transfectants was due to the prolongation of G1 phase. Similar prolongation of G1 phase was observed when BALB/3T3 cells were transferred from 37 to 32°C (6.1 vs. 12.7 h). In addition to prolongation of G1 phase, the duration of S + G2 + M phase was prolonged in BALB/3T3 cells cultured at 32°C, but not in CIRP transfectants cultured at 37°C (Table I). These results indicate that CIRP plays a role in regulation of growth in response to temperature downshifts, but other factors also contribute to the cold-induced growth impairment.

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