Unknown,Transcriptomics,Genomics,Proteomics

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The RNA helicase CrhR as a regulatory factor

ABSTRACT: 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 30M-BM-0C and after 3h of culturing at 20M-BM-0C. Each sample was done in biological triplicates.

ORGANISM(S): Synechocystis sp. PCC 6803

SUBMITTER: Jens Georg

PROVIDER: E-GEOD-58544 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Iron is an essential cofactor in many metabolic reactions. Mechanisms controlling iron homeostasis need to respond to changes in extracellular conditions, but must also keep the concentration of intracellular iron under strict control, as free ferrous iron (Fe2+) can lead to the generation of reactive oxygen species. Due to its role as redox carrier in photosynthesis, the iron quota in cyanobacteria is about 10 times higher than in model enterobacteria, but the molecular details how such high quota is tightly regulated have remained obscure. Here, we measured time-resolved gene expression changes after iron depletion in the cyanobacterium Synechocystis sp. PCC 6803 using a comprehensive microarray platform monitoring both protein-coding and non-coding transcripts. In total, 644 protein-coding genes were differentially expressed during the first 72h. Many of these proteins are associated with iron transport, photosynthesis or ATP synthesis. Comparing our data with three previous studies, we identified a core set of 28 genes involved in the iron stress response. Among them were genes encoding proteins important for assimilation of inorganic carbon, suggesting a previously unknown link between the carbon and iron regulatory networks. Nine of the 28 genes are of unknown function and constitute key targets for detailed functional analysis. Applying identical statistical and clustering criteria as for the protein-coding fraction, we also identified 10 small RNAs, 62 anti-sense RNAs, four 5M-bM-^@M-^YUTRs and 7 intergenic elements as likely to be involved in the iron regulatory network. Hence, our genome-wide profiling results indicate an unprecedented complexity in the iron-related regulatory network of cyanobacteria. We monitored iron limitation stress induced gene expression changes in the cyanobacterial model organism Synechocystis sp. PCC 6803. We included a control sample without iron-stress (0h) and 5 samples from timepoints after stress induction (3h, 12h, 24h, 48h, 72h). Each timepoint was sampled from two independent biological replicates.

Project description:Every cyanobacterial species contains gene encoding site-2-protease (S2P) homolog. The studied prokaryotic S2P homologs play essential roles in regulating stress response through intramembrane proteolysis of membrane-bound anti-sigma factors. Here, gene of Slr0643, one of four S2P homologs in Synechocystis sp. PCC 6803, was insertionally disrupted to explore its physiological role. Only a partially segregated mutant was obtained, indicating its indispensability for growth. The partially disrupted mutant could not grow at pH 6.5, while wild type acclimated to pH 6.5 quickly. The slr0643 gene expression was transiently induced after pH transfer from 7.5 to 6.5. Both evidences demonstrated the pivotal role of fully functional Slr0643 in acid acclimation. DNA microarray and quantitative RT-PCR analyses decoded genes involved in early acid acclimation and revealed differentially expressed genes due to slr0643 disruption at both pH conditions. Early acid acclimation to pH 6.5 included upregulation of sigH, hik16 and hik35, and downregulation of pcrR and sigG; as well as downregulation of porins and upregulation of inorganic carbon and nitrogen transporters. Defective photosynthesis and excess expression of NADH dehydrogenase, together with over upregulation of carbon transporter and repression of nitrogen transporter and metabolism gene contributed to the acid lethality of mutant at pH 6.5. Most interestingly, analysis of microarray data revealed the close relationship between slr0643 disruption and expression of sigH operon. Therefore it was implied that Slr0643/Sll0857/SigH might acts through S2P/anti-Sigma factor/Sigma factor mechanism to play a role in acid acclimation. M-bM-^@M-^C Loop design was used to compare differential expression of wild type and mutant at pH 7.5 and pH 6.5 respectively, including dye-swape. Biological variation was sampled by extracting RNA from three independent experiments and pooling them together before hybridizations. Two or four replicate chips for each comparison and two replicate printings per chip represent technical repeats.

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The RNA helicase CrhR as a regulatory factor

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