Bisaria, N. | Search bioRxiv

bioRxiv2018

The biochemical basis of microRNA targeting efficacy

MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of mRNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA-target affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute-miRNA complexes and all [≤]12-nucleotide sequences. This approach revealed noncanonical target sites unique to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks.

biochemistry↗

bioRxiv2018

RNA tertiary structure energetics predicted by an ensemble model of the RNA double helix

Over 50% of residues within functional structured RNAs are base-paired in Watson-Crick helices, but it is not fully understood how these helices geometric preferences and flexibility might influence RNA tertiary structure. Here, we show experimentally and computationally that the ensemble fluctuations of RNA helices substantially impact RNA tertiary structure stability. We updated a model for the conformational ensemble of the RNA helix using crystallographic structures of Watson-Crick base pair steps. To test this model, we made blind predictions of the thermodynamic stability of >1500 tertiary assemblies with differing helical sequences and compared calculations to independent measurements from a high-throughput experimental platform. The blind predictions accounted for thermodynamic effects from changing helix sequence and length with unexpectedly tight accuracies (RMSD of 0.34 and 0.77 kcal/mol, respectively). These comparisons lead to a detailed picture of how RNA base pair steps fluctuate within complex assemblies and suggest a new route toward predicting RNA tertiary structure formation and energetics.

biophysics↗