Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

Abstract

RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptomewide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.RNA | next-generation sequencing | systems biochemistry | RNA binding proteins | Vts1 R NA-binding proteins (RBPs) constitute 5-10% of the eukaryotic proteome (1-3) and collectively govern the localization, translation, and decay of virtually every transcript (4-6). Despite the ubiquity of RBPs and their central importance in gene regulation, decoding the links between RNA primary sequence and its cadre of regulators remains a major unresolved challenge (7). Current approaches for characterizing RBP function generally involve trade-offs between throughput, comprehensiveness, and quantitative precision. Biophysical measurements can be made with targeted biochemical approaches such as electrophoretic mobility shift assays (EMSAs) or fluorescence polarization (FP) (8, 9), but these methods can only interrogate known RNA-protein interactions and are inherently lowthroughput. Selection-based approaches [e.g., in vitro selection, high-throughput sequencing of RNA, and sequence-specificity landscapes (SEQRS)/RNA bind-n-seq (RBNS)] achieve higher throughput, but these techniques remove binding sites from their natural sequence context and identify "winners" based on more than simple affinity (10). Transcriptome-wide methods, which often use cross-linking and immunoprecipi...

Quantitative high-throughput tests of ubiquitous RNA secondary structure prediction algorithms via RNA/protein binding

“…Recently-developed technologies like RNA-MaP allow the folding of thousands of different RNA sequences to be measured in parallel (19)(20)(21). In RNA-MaP, a library of RNAs is expressed and immobilized within sequence-identified clusters on an Illumina MiSeq chip, and binding of fluorescently-labeled interaction partners is directly monitored across a range of concentrations to determine binding affinity ( Fig.…”

“…RNA-MaP binding measurements were performed using a repurposed Illumina GAIIx instrument with a custom fluidics adapter interface, as described in (19,21). Double-stranded DNA was regenerated on a sequenced Illumina MiSeq flowcell using biotinylated primers.…”

“…The fluorescence data were quantified as described in (21,27). The fluorescence values at each protein concentration, normalized by the amount of transcribed RNA per cluster, were fit to a single-site equilibrium binding equation with a non-specific binding term (27):…”

“…Advances in high-throughput biophysics now make it possible to systematically probe energetics for large numbers of RNAs (19)(20)(21). The RNA-MaP platform allows direct, parallel equilibrium and kinetic measurements of binding of thousands of RNAs with their RNA or protein interaction partners.…”

Ab-initiodiscovery of tumoricidal oligonucleotides in a DNA sequencing machine

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