Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.

Current approaches to design efficient antisense RNAs (asRNAs) rely primarily on a thermodynamic understanding of RNA–RNA interactions. However, these approaches depend on structure predictions and have limited accuracy, arguably due to overlooking important cellular environment factors. In this work, we develop a biophysical model to describe asRNA–RNA hybridization that incorporates in vivo factors using large-scale experimental hybridization data for three model RNAs: a group I intron, CsrB and a tRNA. A unique element of our model is the estimation of the availability of the target region to interact with a given asRNA using a differential entropic consideration of suboptimal structures. We showcase the utility of this model by evaluating its prediction capabilities in four additional RNAs: a group II intron, Spinach II, 2-MS2 binding domain and glgC 5΄ UTR. Additionally, we demonstrate the applicability of this approach to other bacterial species by predicting sRNA–mRNA binding regions in two newly discovered, though uncharacterized, regulatory RNAs.

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Ribonucleoproteins (RNPs) are vital to many cellular events. To this end, many neurodegenerative diseases and cancers have been linked to RNP malfunction, particularly as this relates to defective processing of cellular RNA. The connection of RNPs and diseases has also propagated a shift of focus onto RNA targeting from traditional protein targeting treatments. However, therapeutic development in this area has been limited by incomplete molecular insight into the specific contributions of RNPs to disease. This review outlines the role of several RNPs in diseases, focusing on molecular defects in processes that affect proper RNA handling in the cell. This work also evaluates the contributions of recently developed methods to understanding RNP association and function. We review progress in this area by focusing on molecular malfunctions of RNPs associated with the onset and progression of several neurodegenerative diseases and cancer and conclude with a brief discussion of RNA-based therapeutic efforts.

Fluorescence-based tools that measure RNA-RNA and RNA-protein interactions in vivo offer useful experimental approaches to probe the complex and dynamic physiological behavior of bacterial RNAs. Here we document the step-by-step design and application of two fluorescence-based methods for studying the regulatory interactions RNAs perform in vivo: (i) the in vivo RNA Structural Sensing System (iRS) for measuring RNA accessibility and (ii) the trifluorescence complementation (TriFC) assay for measuring RNA-protein interactions.