Conserved mechanisms for cold adaptation in bacteria and higher plants

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Cold shock domain (CSD) protein or Y-box protein is widely distributed in bacteria, plants, and animals. The highly conserved CSD serves as a nucleic acid-binding domain for the functions in translational and/or transcriptional regulations within these proteins. We have identified a cDNA clone from winter wheat (WCSP1) which encodes a protein homologous to E.. coli CspA. The putative WCSP1 protein consists of an N-terminal CSD and three C-terminal CCHC zinc fingers. Both WCSP1 mRNA and protein levels steadily increases in crown tissue during cold acclimation. Recombinant WCSP1 protein is capable of binding RNA and ss/dsDNA. WCSP1 partially complemented a cold sensitive phenotype of E.coli cspA, cspB, cspE, cspG quadruple mutant, suggesting that WCSP1 shares a function with E.coli CSPs for cold adaptation. It has also been demonstrated that WCSP1 showed in vivo RNA chaperone activity in E.coli system. A series of deletion and point mutations were introduced in WCSP1 and the mutant recombinant proteins were purified from E.coli. ssDNA-binding assay revealed that the C-terminal Gly-rich and zinc finger region is not required for ssDNA binding. Point mutations in the RNP I and RNP II RNA recognition motifs within the CSD abolished ssDNA-binding activity. A molecular beacon system with 5'-FITC-labeled and 3'-quencher-labeled oligonucleotides was utilized to demonstrate a double strand nucleic acid melting activity in vitro. The melting activity of WCSP1 and its mutant proteins correlated well with ssDNA binding activity. Therefore it was concluded that RNA chaperone activity of WCSP1 resides within the CSD. Transient expression of WCSP1-GFP protein revealed that WCSP1 is localized in ER and nucleus. The ER localization required the C-terminal zinc finger domain of WCSP1.