The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts

Abstract

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.

AtpΘ is an inhibitor of F0F1 ATP synthase to arrest ATP hydrolysis during low-energy conditions in cyanobacteria

“…CuSO 4 (2 μM) was used to induce the expression of the Cu 2+ -responsive petE promoter 43 , while the petJ promoter was induced by removing Cu 2+ from the medium through centrifugation and resuspension. For high-density cultivation used for ATP synthase purification, Synechocystis 6803 overexpressing P petE -atpB-3xFLAG was cultured in the cell-DEG system as reported previously 44 using freshwater medium 45 with the following modifications: Na 2 EDTA and CuSO 4 were not included in the medium, and 10 µg/mL kanamycin or 5 µg/mL gentamicin was added.…”