Cotranscriptional RNA folding is widely assumed to influence the timely control of gene expression, but our understanding remains limited. In bacteria, the fluoride (F−)-sensing riboswitch is a transcriptional control element essential to defend against toxic F− levels. Using this model riboswitch, we find that its ligand F− and essential bacterial transcription factor NusA compete to bind the cotranscriptionally folding RNA, opposing each other’s modulation of downstream pausing and termination by RNA polymerase. Single-molecule fluorescence assays probing active transcription elongation complexes discover that NusA unexpectedly binds highly reversibly, frequently interrogating the complex for emerging, cotranscriptionally folding RNA duplexes. NusA thus fine-tunes the transcription rate in dependence of the ligand-responsive higher-order structure of the riboswitch. At the high NusA concentrations found intracellularly, this dynamic modulation is expected to lead to adaptive bacterial transcription regulation with fast response times.

Here, we introduce CS-Annotate, a tool that uses assigned NMR chemical shifts to annotate structural features in RNA. At its core, CS-Annotate is a deployment of a multitask deep learning model that simultaneously classifies the solvent exposure, base-stacking and -pairing status, and conformation of individual RNA residues from their chemical shift fingerprint. Here, we briefly describe how we trained and tested the classifier and demonstrate its application to a model RNA system. CS-Annotate can be accessed via the SMALTR (Structure-based MAchine Learning Tools for RNA) Science Gateway (smaltr.org).

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, associated with various forms of cognitive and functional impairment which worsen with disease progression. AD is typically characterized as a protein misfolding disease, in which abnormal plaques form due to accumulation of tau and b-amyloid (Ab) proteins. In the latter case, amyloid precursor protein (APP)a transmembrane protein critical for neuron growth and survival -is proteolytically cleaved by an assortment of proteins into small peptides which accumulate into Ab plaques. Among the proteins involved in this process, sortilin-related receptor 1 (SORL1) is believed to be involved in APP processing and trafficking. Recently, a genome-wide study of microRNA-related variants found that a single nucleotide polymorphism (SNP) within premature microRNA-1229 (pre-miR-1229) is significantly associated with AD. This particular SNP causes altered processing of pre-miR-1229 via the microRNA pathway, resulting in an increased production of miR-1229-3p, a regulator of SORL1 translation and expression. Here, we show that the wild-type (WT) pre-miR-1229 forms a G-quadruplex structure in equilibrium with the long, extended hairpin structure. Moreover, we hypothesized that the AD-associated SNP within pre-miR-1229 causes a shift in secondary structure equilibrium from G-quadruplex to hairpin, resulting in increased recognition and processing by the endonuclease Dicer in the micro-RNA pathway. To determine this, we utilized various biophysical techniques, such as NMR spectroscopy, CD spectroscopy, and UV thermal denaturation experiments to characterize both WT-and SNP-associated secondary structures within pre-miR-1229. Herein, we propose a mechanism by which the SNP within pre-miR-1229 results in increased production of its mature form which further targets SORL1 mRNA and inhibits its translation.

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