Global Mapping of Hfq-dependent RNA-RNA interactions in Caulobacter crescentus

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Post-transcriptional control by small, regulatory RNAs (sRNAs) is a major layer of gene expression regulation in bacteria. The advent of high-throughput sequencing technologies has revealed hundreds of sRNA candidates in different bacterial species, however, their functional characterization is lacking behind. Most sRNAs act through inter-molecular base-pairings with trans-encoded mRNAs, leading to changes in stability, processing and/or translation of these transcripts. In Gram-negative bacteria, these short, often imperfect interactions between RNAs are typically mediated by the highly conserved RNA chaperone, Hfq. This project proposal aims at the discovery of Hfq-dependent RNA-RNA interactions in the model alpha-proteobacterium, Caulobacter crescentus. Our preliminary data revealed dozens of Hfq-binding sRNAs in C. crescentus, however, how these add to the lifecycle and physiology of this organism is currently unknown. Here, we propose to employ RIL-seq (RNA interaction by ligation and sequencing) to map the landscape Hfq-mediated RNA duplex formation in C. crescentus at a global scale. In contrast to other methods, RIL-seq is able to reveal unconventional interaction partners of sRNAs, including sponge RNAs. Application of RIL-seq to C. crescentus is expected to not only expand the knowledge on the interaction partners of distinct sRNAs, but also to uncover novel mechanistic concepts of RNA-based regulation in this organism.