Wolin Erica, Guo Jimmy K, Blanco Mario R, Goronzy Isabel N, Gorhe Darvesh, Dong Wenzhao, Perez Andrew A, Keskin Abdurrahman, Valenzuela Elizabeth, Abdou Ahmed A, Urbinati Carl R, Kaufhold Ross, Rube H Tomas, Brito Querido Jailson, Guttman Mitchell, Jovanovic Marko
Department of Biological Sciences, Columbia University, New York City, NY 10027, USA.
Division of Biology and Bioengineering, California Institute of Technology, Pasadena, CA 91125, USA; Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
Cell. 2025 Jul 22. doi: 10.1016/j.cell.2025.06.042.
RNA-binding proteins (RBPs) regulate all stages of the mRNA life cycle, yet current methods generally map RNA targets of RBPs one protein at a time. To overcome this limitation, we developed SPIDR (split-and-pool identification of RBP targets), a highly multiplexed split-pool method that profiles the binding sites of dozens of RBPs simultaneously. SPIDR identifies precise, single-nucleotide binding sites for diverse classes of RBPs. Using SPIDR, we uncovered an interaction between LARP1 and the 18S rRNA and resolved this interaction to the mRNA entry channel of the 40S ribosome using cryoelectron microscopy (cryo-EM), providing a potential mechanistic explanation for LARP1's role in translational suppression. We explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 preferentially associates with translationally repressed mRNAs upon mTOR inhibition. SPIDR has the potential to significantly advance our understanding of RNA biology by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.
RNA结合蛋白（RBPs）调控着mRNA生命周期的各个阶段，但目前的方法通常一次只能对一种蛋白的RNA靶标进行定位。为克服这一局限性，我们开发了SPIDR（RBP靶标的拆分合并鉴定法），这是一种高度多重的拆分合并方法，可同时分析数十种RBPs的结合位点。SPIDR能为不同类别的RBPs鉴定出精确的单核苷酸结合位点。利用SPIDR，我们发现了LARP1与18S rRNA之间的相互作用，并通过冷冻电子显微镜（cryo-EM）将这种相互作用解析到40S核糖体的mRNA进入通道，为LARP1在翻译抑制中的作用提供了潜在的机制解释。我们研究了mTOR抑制后RBP结合的变化，发现mTOR抑制后4EBP1优先与翻译受抑制的mRNA结合。SPIDR有潜力通过以前所未有的规模快速、从头发现RNA-蛋白质相互作用，显著推进我们对RNA生物学的理解。
