A DEAD-box RNA helicase produces two forms of transcript that differentially respond to cold stress in a cryophyte (Chorispora bungeana).

ABSTRACT

MAIN CONCLUSION: This work demonstrated that a cold-induced DEAD-box RNA helicase, CbDRH, is also post-transcriptionally regulated upon cold stress, and it interacts with a cold-responsive, glycine-rich, RNA-binding protein, CbGRP. Chorispora bungeana (C. bungeana) is a representative alpine subnival plant species that shows strong tolerance to multiple abiotic stresses, especially cold stress. DEAD-box RNA helicases are implicated in almost all RNA metabolic processes and participate in multiple abiotic stress responses. Here, we characterized a cold-induced DEAD-box RNA helicase gene from C. bungeana. We cloned the full-length cDNA of the gene by RACE and called it C. bungeana DEAD-box RNA Helicase (CbDRH). Structurally, CbDRH possesses all nine conserved motifs characteristic of DEAD-box protein family members in its central region, and the N- and C- terminal extensions both harbor a glycine-rich region containing several RGG-box motifs. The CbDRH gene produces two forms of transcripts, CbDRH.2 and CbDRH.1, by alternative splicing. CbDRH.2 comes from the complete excision of all the nine introns, while CbDRH.1 results from the use of an alternative 5' splice site in the eighth intron, retaining part of the intron (the first 260 bp) with an early stop codon. Semi-quantitative RT-PCR analysis showed that CbDRH.2, but not CbDRH.1, is up-regulated by cold stress. However, the abundance of CbDRH.1 transcript can be elevated by cycloheximide (an inhibitor of nonsense-mediated decay) treatment, indicating that CbDRH.1 is targeted to nonsense-mediated decay (NMD). A subcellular localization analysis showed that CbDRH.2 protein is located in the nuclei. Further investigation suggested that CbDRH.2 can interact with a cold-responsive, glycine-rich, RNA-binding protein, CbGRP (Chorispora bungeana glycine-rich, RNA-binding protein). These data suggest that the cold-induced CbDRH is also post-transcriptionally regulated under cold stress and that CbDRH.2 may function together with the glycine-rich, RNA-binding protein, CbGRP, in the cold stress response.