The RNA helicase CrhR as a regulatory factor

DEAD-box RNA-helicases catalyze the correct folding of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083) whose inactivation has severe effects on cellular physiology and morphology, particularly under cold stress. In order to identify potential RNA targets of CrhR, custom microarrays containing sequences corresponding to non-coding small RNAs (UTRs, ncRNAs, asRNAs, iRNAs) and all mRNAs were constructed. The RNAs exhibiting CrhR-dependent alteration of expression constitute both mRNA and small RNA transcripts. CrhR therefore is not a global regulator of RNA metabolism but instead interacts with a specific subset of the total RNA repertoire in Synechocystis, a CrhR-specific RNA regulon that functions predominately at low temperature. RNA half-life analyses indicated that expression of regulon members is controlled by a mechanism that does not involve CrhR-dependent RNA turnover. Biochemically, CrhR is able to anneal but not unwind an asRNA-mRNA target in vitro, providing evidence that regulation occurs through an RNA duplex intermediate. The data reveal a novel mechanism regulating the differential expression of operon members, crhR mutation disrupts a negative regulatory feedback loop involving CrhR regulation at an early stage of transcription in affected operons. We propose that CrhR is functioning as a RNA chaperone, similar to Hfq in enterobacteria, but regulating both post-transcriptional events and transcription termination, by altering sRNA metabolism. We monitored gene expression of an Synechocystis PCC6083 WT and a partial crhR knockout at 30°C and after 3h of culturing at 20°C. Each sample was done in biological triplicates.