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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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RNA-binding property of CIRP. (a) Bacterially  expressed GST protein and  GST–CIRP fusion protein  demonstrated by Coomassie  blue staining. (b) Northwestern blots showing binding of  GST and GST–CIRP proteins  to radiolabeled ribonucleotide homopolymers, poly(A),  poly(C), poly(G), and poly(U),  at different salt concentrations as indicated.
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Figure 4: RNA-binding property of CIRP. (a) Bacterially expressed GST protein and GST–CIRP fusion protein demonstrated by Coomassie blue staining. (b) Northwestern blots showing binding of GST and GST–CIRP proteins to radiolabeled ribonucleotide homopolymers, poly(A), poly(C), poly(G), and poly(U), at different salt concentrations as indicated.

Mentions: We expressed and purified CIRP as a GST–fusion protein (Fig. 4 a) and analyzed its RNA-binding activity by Northwestern blot assay (Matunis et al., 1992; Schumacher et al., 1995). The GST–CIRP protein bound to all RNA homopolymers to a varying extent at low NaCl concentrations, while control GST did not (Fig. 4 b). At high NaCl concentrations, GST–CIRP bound only to poly(U), suggesting that CIRP protein possessed a specific RNA-binding activity.


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)

RNA-binding property of CIRP. (a) Bacterially  expressed GST protein and  GST–CIRP fusion protein  demonstrated by Coomassie  blue staining. (b) Northwestern blots showing binding of  GST and GST–CIRP proteins  to radiolabeled ribonucleotide homopolymers, poly(A),  poly(C), poly(G), and poly(U),  at different salt concentrations as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: RNA-binding property of CIRP. (a) Bacterially expressed GST protein and GST–CIRP fusion protein demonstrated by Coomassie blue staining. (b) Northwestern blots showing binding of GST and GST–CIRP proteins to radiolabeled ribonucleotide homopolymers, poly(A), poly(C), poly(G), and poly(U), at different salt concentrations as indicated.
Mentions: We expressed and purified CIRP as a GST–fusion protein (Fig. 4 a) and analyzed its RNA-binding activity by Northwestern blot assay (Matunis et al., 1992; Schumacher et al., 1995). The GST–CIRP protein bound to all RNA homopolymers to a varying extent at low NaCl concentrations, while control GST did not (Fig. 4 b). At high NaCl concentrations, GST–CIRP bound only to poly(U), suggesting that CIRP protein possessed a specific RNA-binding activity.

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