By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions. We identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of 'cross-linking and immunoprecipitation' (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (χCRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the χCRAC method and presents recent results obtained with the Nrd1 RBP. 1. This method utilizes ultraviolet (UV, 254 nm) irradiation to induce covalent bonds between RBPs and their directly bound RNAs (i.e., zero-distance cross-linking). Subsequently, the RBP of interest is immunopurified under stringent conditions to ensure that only RNAs covalently cross-linked to the proteins are identified. Bound RNAs are then partially digested with RNases and subsequently converted into cDNA libraries for sequencing. The high purification stringency is important as it greatly increases the specificity of protein and RNA recovery, which is also further enhanced through SDS-PAGE purification of the cross-linked ribonucleoprotein (RNP) complex. CLIP and related methods also provide nucleotide resolution insight into the protein binding site, because during the preparation of the sequencing library, amino acids that cross-linked to the RNA frequently terminate the reverse transcriptase or cause the enzyme to introduce mutations at this site 1,2,3 . Since its introduction, the original CLIP protocol has produced a remarkable variety of derivative methodologies. A particularly important breakthrough came with the development of HITS-CLIP (or CLIP-seq), which merges high-throughput sequencing with the CLIP approach 3 . This has since been adopted by all CLIP-based methodologies. iCLIP introduced improvements in the RNase-mediated trimming and adaptor ligation techniques that facilitate more accurat...

27By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to very 28 efficiently adapt to constant changes in their environment. To better understand how 29 Escherichia coli acclimatizes to changes in nutrient availability, we performed UV cross-30 linking, ligation and sequencing of hybrids (CLASH) to uncover sRNA-target interactions. 31Strikingly, we uncovered hundreds of novel Hfq-mediated sRNA-target interactions at specific 32 growth stages, involving many novel 3'UTR-derived sRNAs and a plethora of sRNA-sRNA 33 interactions. We discovered sRNA-target interaction networks that play a role in adaptation to 34 changes in nutrient availability. We characterized a novel 3'UTR-derived sRNA (MdoR), which 35 is part of a regulatory cascade that enhances maltose uptake by (a) inactivating repressive 36 pathways that block the accumulation of maltose transporters and (b) by reducing the flux of 37 general porins to the outer membrane. Our work provides striking examples of how bacteria 38 utilize sRNAs to integrate multiple regulatory pathways to enhance nutrient stress adaptation.39 3 Microorganisms are renowned for their ability to adapt to environmental changes by 40 rapidly rewiring their gene expression program. These responses are mediated through 41 integrated transcriptional and post-transcriptional networks. Control at the transcriptional level 42 dictates which genes are expressed (Balleza et al., 2009; Martínez-Antonio et al., 2008) and 43 is well-characterised in Escherichia coli. Post-transcriptional regulation is key for controlling 44 adaptive responses. By using riboregulators and RNA-binding proteins (RBPs), cells can 45 efficiently integrate multiple pathways and incorporate additional signals into regulatory 46 circuits. E. coli employs many post-transcriptional regulators, including small regulatory RNAs 47 (sRNAs (Waters and Storz, 2009)), cis-acting RNAs (Kortmann and Narberhaus, 2012), and 48 RNA binding proteins (RBPs) (Holmqvist and Vogel, 2018). The sRNAs are the largest class 49 of bacterial regulators, which work in tandem with RBPs to regulate their RNA targets (Storz 50 et al., 2011; Waters and Storz, 2009). The base-pairing interactions are often mediated by 51 RNA chaperones such as Hfq and ProQ, which help to anneal or stabilize the sRNA and 52 sRNA-target duplex (Smirnov et al., 2017(Smirnov et al., , 2016 Updegrove et al., 2016). Small RNAs can 53 repress or stimulate translation and transcription, as well as control mRNA stability 54 (Sedlyarova et al., 2016; Updegrove et al., 2016; Vogel and Luisi, 2011; Waters and Storz, 55 2009). 56During growth in rich media, E. coli are exposed to continuously changing conditions, 57 such as fluctuations in nutrient availability, pH and osmolarity. Consequently, E. coli elicit 58 complex responses that result in physiological and behavioural changes such as envelope 59 composition remodelling, quorum sensing, nutrient scavenging, swarming and biofilm 60 formation. Even subtle changes in the growth conditions can trigg...