Cyanobacteria occupy very diverse habitats with rapidly changing environmental conditions, which forces them to develop effective response mechanisms in order to survive. Post-transcriptional control of gene expression, which is mostly determined by the function of regulatory RNA molecules and the RNA degradation apparatus, provides an important mechanism for adaptation to environmental demands. Investigation of major players in RNA degradation and maturation in the model cyanobacterium Synechocystis sp. PCC6803, namely homologs of RNase E/G (Rne) and RNase III (Rnc2), was the main focus of the present work. As RNA chaperone Hfq, which facilitates otherwise imperfect sRNA-mRNA base pairing, functions as a post-transcriptional regulator of gene expression in many bacteria, we also studied two Hfq-&
#8208;dependent sRNAs Hpr8 and Hpr10 with a closer look on their degradation patterns.In order to clarify protein-RNA interactions between studied RNases and their possible RNA targets in vivo a genome wide analysis of binding sites for Rne and Rnc2 was performed using individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) combined with Solexa high-throughput sequencing. This novel approach confirmed that Rne binds to the stem loop structure in the 5 UTR of rne gene and therefore most likely regulates its own synthesis in a similar manner as it has been shown for E. coli. Discovery of Rne binding sites within the rRNA precursor between 23S and 5S rRNAs led to the assumption that the maturation of 5S rRNA in Synechocystis is analogous to the one in E. coli. Conducted in vitro cleavage assays and a 3 RACE experiment substantiated this hypothesis and proved the accuracy of results provided by iCLIP method. We also revealed interaction of Rne with a number of sRNAs. In vitro cleavage assays were performed to verify Rne-dependent processing of some of the putative targets. Interestingly, we could see a clear pattern in Rne interaction with tRNAs: analysis of the location of the binding site determined that Rne always binds to the anticodon loop of tRNAs; an additional binding site at the variable loop of some tRNAs was also discovered.Evaluation of Rnc2 binding properties was completed by implementing iCLIP approach as well. Detection of Rnc2 binding sites within rRNAs and tRNAs suggested involvement of this RNase in maturation of their precursors in Synechocystis as it has been shown for other bacteria. We could also observe that the two studied RNases Rne and Rnc2 in some cases have binding sites mapped to the same transcripts and therefore might act together. In addition we could demonstrate using in vitro cleavage assays that the sRNA Hpr10 is a true substrate for Rnc2. iCLIP experiment revealed a binding site next to a long double-stranded region within this sRNA, where processing most likely occurs. In summary, we could show that the iCLIP method can be used for the study of RNase-RNA interactions in bacteria. Verification of iCLIP data using in vitro assays confirmed that several RNAs are true targets of the respective RNases. Clearly, more comprehensive studies are needed in the future to analyse the specific functions of these ribonucleases in post-&
#8208;transcriptional gene regulation.
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