Nugent Patrick J, Park Heungwon, Wladyka Cynthia L, Yelland James N, Sinha Sayantani, Chen Katharine Y, Bynum Christine, Quarterman Grace, Lee Stanley C, Hsieh Andrew C, Subramaniam Arvind Rasi
Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA.
Nat Methods. 2025 May 29. doi: 10.1038/s41592-025-02702-6.
RNAs undergo a complex choreography of metabolic processes that are regulated by thousands of RNA-associated proteins. Here we introduce ReLiC, a scalable and high-throughput RNA-linked CRISPR approach to measure the responses of diverse RNA metabolic processes to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC relies on an iterative strategy to integrate genes encoding Cas9, single-guide RNAs (sgRNAs) and barcoded reporter libraries into a defined genomic locus. Combining ReLiC with polysome fractionation reveals key regulators of ribosome occupancy, uncovering links between translation and proteostasis. Isoform-specific ReLiC captures differential regulation of intron retention and exon skipping by SF3B complex subunits. Chemogenomic ReLiC screens decipher translational regulators upstream of messenger RNA (mRNA) decay and identify a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a powerful framework for discovering and dissecting post-transcriptional regulatory networks in human cells.
RNA会经历由数千种RNA相关蛋白调控的复杂代谢过程编排。在此，我们介绍了ReLiC，这是一种可扩展的高通量RNA连接CRISPR方法，用于测量多种RNA代谢过程对2092个编码所有已知RNA相关蛋白的人类基因敲除的反应。ReLiC依靠一种迭代策略，将编码Cas9、单向导RNA（sgRNA）和条形码报告文库的基因整合到一个确定的基因组位点。将ReLiC与多核糖体分级分离相结合，揭示了核糖体占据的关键调节因子，揭示了翻译与蛋白质稳态之间的联系。异构体特异性ReLiC捕获了SF3B复合亚基对内含子保留和外显子跳跃的差异调节。化学基因组学ReLiC筛选揭示了信使RNA（mRNA）衰变上游的翻译调节因子，并确定了核糖体碰撞传感器GCN1在抗白血病药物高三尖杉酯碱治疗期间的作用。我们的工作证明ReLiC是发现和剖析人类细胞中转录后调控网络的强大框架。
