• Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing.

      Chihara, Kotaro; Bischler, Thorsten; Barquist, Lars; Monzon, Vivian A; Noda, Naohiro; Vogel, Jörg; Tsuneda, Satoshi (2019-12-03)
      Bacterial small noncoding RNAs (sRNAs) play posttranscriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the Pseudomonas aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5' untranslated regions (UTRs) and sRNAs and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that the association of sRNAs with Hfq is primarily a function of their expression levels, strongly supporting the notion that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of posttranscriptional regulation in P. aeruginosaIMPORTANCE The Gram-negative bacterium P. aeruginosa is ubiquitously distributed in diverse environments and can cause severe biofilm-related infections in at-risk individuals. Although the presence of a large number of putative sRNAs and widely conserved RNA chaperones in this bacterium implies the importance of posttranscriptional regulatory networks for environmental fluctuations, limited information is available regarding the global role of RNA chaperones such as Hfq in the P. aeruginosa transcriptome, especially under different environmental conditions. Here, we characterize Hfq-dependent differences in gene expression and biological processes in two physiological states: the planktonic and biofilm forms. A combinatorial comparative CLIP-seq and total RNA-seq approach uncovered condition-dependent association of RNAs with Hfq in vivo and expands the potential direct regulatory targets of Hfq in the P. aeruginosa transcriptome.
    • Plugging Small RNAs into the Network.

      Barquist, Lars; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (ASM, 2020-06-02)
      Small RNAs (sRNAs) have been discovered in every bacterium examined and have been shown to play important roles in the regulation of a diverse range of behaviors, from metabolism to infection. However, despite a wide range of available techniques for discovering and validating sRNA regulatory interactions, only a minority of these molecules have been well characterized. In part, this is due to the nature of posttranscriptional regulation: the activity of an sRNA depends on the state of the transcriptome as a whole, so characterization is best carried out under the conditions in which it is naturally active. In this issue of mSystems, Arrieta-Ortiz and colleagues (M. L. Arrieta-Ortiz, C. Hafemeister, B. Shuster, N. S. Baliga, et al., mSystems 5:e00057-20, 2020, https://doi.org/10.1128/mSystems.00057-20) present a network inference approach based on estimating sRNA activity across transcriptomic compendia. This shows promise not only for identifying new sRNA regulatory interactions but also for pinpointing the conditions in which these interactions occur, providing a new avenue toward functional characterization of sRNAs.