Role of small regulatory RNA networks in controlling adaptive responses in Escherichia coli

Microorganisms are exposed to constantly changing environments, and consequently have evolved mechanisms to rapidly adapt their physiology upon stress imposition. These adaptive responses are coordinated through the rewiring of gene expression via complex networks that control the transcriptional program and the activity of post-transcriptional regulators. Although transcription factors primarily determine which genes are expressed, post-transcriptional regulation has a major role in fine-tuning the dynamics of gene expression. Post-transcriptional control is exerted by RNA-binding proteins and small regulatory RNAs (sRNAs) that bind to mRNA targets and modulate their synthesis, degradation and translation efficiency. In Escherichia coli, sRNAs associated with an RNA chaperone, Hfq, are key post-transcriptional regulators, yet the functions of most of these sRNAs are still unknown. The first step in understanding the roles of sRNAs in regulating gene expression is to identify their targets. To generate transcriptome-wide maps of Hfq-mediated sRNA-mRNA binding, we applied CLASH (cross-linking, ligation and sequencing of hybrids), a method that combines in vivo capture of RNA-RNA interactions, high-throughput sequencing and computational analyses, in E. coli. We uncovered thousands of dynamic growth-stage dependent association of Hfq to sRNAs and mRNAs. The latter confirmed known sRNA-target pairs and identified additional targets for known sRNAs, as well as novel sRNAs in various genomic features along with their targets. These data significantly expand our knowledge of the sRNA-target interaction networks in E.coli. In particular, the Hfq CLASH data indicated 3’-UTRs of mRNAs as major reservoirs of sRNAs, and the utilization of these may be more common than anticipated. Our findings also provide mechanistic insights that ensue from the identification of tens of sRNA-sRNA interactions that point to extensive sponging activity among regulatory RNAs: many sRNAs appear to be able to interact and repress the functions of other base-pairing sRNAs. We validated and highlighted the biological significance of some of the CLASH results by characterizing a 3’-UTR derived sRNA, MdoR (mal-dependent OMP repressor). This sRNA emerges by processing of the last transcript of malEFG polycistron, encoding components of maltose transport system. We found MdoR directly downregulates several major porins, whilst derepressing the maltose-specific porin LamB via destabilization of its inhibitor, MicA, likely by a sponging mechanism. Physiologically, MdoR contributes to the remodelling of envelope composition and links nutrient sensing to envelope stress responses during maltose assimilation. MdoR is a clear example of how cells integrate circuitry through multiple networks as part of their adaptive responses and how the CLASH methodology can help expand our understanding of sRNA-based regulation

A computationally-enhanced hiCLIP atlas reveals Staufen1-RNA binding features and links 3′ UTR structure to RNA metabolism

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3' UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3' UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions

Motivations for Using Social Networking Sites: The Case of Romania

Social networking sites (SNS) have been experiencing a growth boom in latter years, consequently drawing marketers’ attention as an important medium for meaningful interaction with the consumer. But achieving brand effectiveness on SNS begins with understanding users’ motivations for online social networking. This paper sought to explore Romanian users’ motivations, and reports findings from a qualitative study consisting of face-to-face in-depth interviews and participant observation. We determine the main differences in motivations for using four major Romanian social networking sites. Implications in terms of brand communication within the context of SNS are also discussed

Patterns of social influence in social networking sites – a design perspective

Social networking sites (SNSs) are quickly becoming the main venues for social interaction of Internet users. As the competition between these online services is growing ever stronger, the success of SNSs derives from persuasive core application designs that influence users in taking specific actions, such as joining the community or recruiting new users. Therefore there is a need to identify relevant, value-adding features that facilitate social interaction and that are integrated in the group’s socialization rituals. The present study employed Cialdini’s framework in the analysis of SNSs design characteristics that facilitate social influence leading to the fulfillment of site marketing objectives, such as member acquisition, member retention and user loyalty. We examined social application features of the three major Romanian SNSs, i.e. Facebook, Hi5 and Neogen on six dimensions corresponding to Cialdini’s social influence principles: reciprocation, consistency, social proof, liking, authority and scarcity. We thereby extract core application commonalities and differences and identify design gaps from a social influence perspective that are to be explored and exploited as a source of competitive advantage. Marketing implications of the results are discussed

Safety Considerations and Proposed Workflow for Laboratory-Scale Chemical Synthesis by Ball Milling

Chemical reactions that take place in a ball mill and in the absence of a bulk reaction solvent present different safety profiles to stirred solution reactions. Herein, we present and describe steps that a researcher may take to better ensure that they have considered some of the hazards and measures that emerge and minimize the risk to themselves and their colleagues

Calcium induced calcium release during action potential firing in developing inner hair cells

In the mature auditory system inner hair cells (IHCs) convert sound induced vibrations into electrical signals that are relayed to the CNS via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarisation, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, inter-spike interval or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behaviour is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data demonstrate that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signalling mechanisms involved in early developmental processes

A computationally-enhanced hiCLIP atlas reveals Staufen1-RNA binding features and links 3′ UTR structure to RNA metabolism

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3′ UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3′ UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions