She R, Chakravarty AK, Layton CJ, Chircus LM, Andreasson JO, Damaraju N, McMahon PL, Buenrostro JD, Jarosz DF, Greenleaf WJ. 2017. Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome. Proceedings of the National Academy of Sciences of the United States of America. 114(14):3619-3624. Pubmed: 28325876 DOI:10.1073/pnas.1618370114


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 transcriptome-wide 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 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 Δ 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.

Related Faculty

Photo of Jason Buenrostro

The Buenrostro lab is broadly dedicated to advancing our knowledge of gene regulation and the downstream consequences on cell fate decisions.

Search Menu