Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome.
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 theSaccharomyces cerevisiaegenome. 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 invts1Δ 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.
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305.
Richard
R
She
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305.
Anupam K
AK
Chakravarty
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.
Curtis J
CJ
Layton
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305.|Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.
Lauren M
LM
Chircus
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305.
Johan O L
JO
Andreasson
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.
Nandita
N
Damaraju
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.|Department of Applied Physics, Stanford University, Stanford, CA 94305.
Peter L
PL
McMahon
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.
Jason D
JD
Buenrostro
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305; wjg@stanford.edu jarosz@stanford.edu.|Department of Developmental Biology, Stanford University, Stanford, CA 94305.
Daniel F
0000-0003-3497-5888
DF
Jarosz
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305; wjg@stanford.edu jarosz@stanford.edu.|Department of Applied Physics, Stanford University, Stanford, CA 94305.
William J
WJ
Greenleaf
D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D053263:Gene Regulatory Networks; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D020871:RNA Stability; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C508663:Vts1 protein, S cerevisiae
RNA; RNA binding proteins; Vts1; next-generation sequencing; systems biochemistry