RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.National Science Foundation (U.S.) (0821391

Understanding microRNA targeting with high-throughput biochemistry

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February, 2021Cataloged from the official PDF of thesis. Vita.Includes bibliographical references.MicroRNAs (miRNAs) are short RNAs that, in complex with Argonaute (AGO) proteins, guide repression of mRNA targets. miRNAs negatively regulate most mammalian mRNAs, and disruption of this regulation often results in severe defects at the cellular and organismal level. miRNA repression occurs primarily through base-pairing between the miRNA seed region (nucleotides 2-8) and mRNA 3'-UTR sites, leading to transient recruitment of mRNA-destabilizing factors. However, only a small fraction of the gene-expression changes caused by a miRNA can currently be predicted, which precludes a deeper understanding of how miRNA regulation impacts the animal transcriptome. miRNA targeting efficacy should in principle be a function of the affinity between AGO-miRNA complexes and their targets. However, only a few such measurements had been reported, with measured values differing from those predicted for RNA-RNA pairing in solution.We therefore adapted a high-throughput biochemical platform utilizing random-sequence RNA libraries to obtain the vast quantity of affinity values required to predict miRNA targeting efficacy. Through a novel analytical approach, we assigned relative dissociation (K[subscript D]) constants to all binding sites </-12 nt in length, for six miRNAs. These analyses revealed unanticipated miRNA-specific differences in the affinity of similar sites, unique sites for different miRNAs, and a 100-fold influence of flanking dinucleotide context surrounding a site. These measurements informed a biochemical model of miRNA targeting that outperformed all existing models of miRNA targeting, which was extended to all miRNAs using a convolutional neural network (CNN) trained on both affinity and repression data. We also applied this high-throughput biochemical approach to understand the role of the miRNA 3' region using partially random RNA libraries.We found unique 3'-pairing preferences for each miRNA, and evidence for two distinct binding modes. The miRNA-specific differences and two binding modes depended on G nucleotides in the miRNA 3' region, thus providing a heuristic by which to extend these findings to target prediction in vivo. This work establishes high-throughput biochemistry combined with mathematical modeling and deep learning as a powerful paradigm for building quantitative models of gene regulation, which might aid in eventually building a complete model of the cell.by Sean E. McGeary.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biolog

Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing

To use microRNAs to downregulate mRNA targets, cells must first process these ∼22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.National Institutes of Health (U.S.) (Grant GM067031)National Institutes of Health (U.S.) (Grant T32GM007753

Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Keywords: competing endogenous RNA; miRNA; target abundance; cooperatively; gene regulation; base pair complementarity; miRNA degradationNational Institutes of Health (U.S.) (Grant GM067031

mRNA Destabilization Is the Dominant Effect of Mammalian MicroRNAs by the Time Substantial Repression Ensues

MicroRNAs (miRNAs) regulate target mRNAs through a combination of translational repression and mRNA destabilization, with mRNA destabilization dominating at steady state in the few contexts examined globally. Here, we extend the global steady-state measurements to additional mammalian contexts and find that regardless of the miRNA, cell type, growth condition, or translational state, mRNA destabilization explains most (66%- > 90%) miRNA-mediated repression. We also determine the relative dynamics of translational repression and mRNA destabilization for endogenous mRNAs as a miRNA is induced. Although translational repression occurs rapidly, its effect is relatively weak, such that by the time consequential repression ensues, the effect of mRNA destabilization dominates. These results imply that consequential miRNA-mediated repression is largely irreversible and provide other insights into the nature of miRNA-mediated regulation. They also simplify future studies, dramatically extending the known contexts and time points for which monitoring mRNA changes captures most of the direct miRNA effects

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium Noncoding RNAs: Biology and Applications brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications