Genome-scale exon perturbation screens uncover exons critical for cell fitness,Molecular Cell

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Genome-scale exon perturbation screens uncover exons critical for cell fitness
Molecular Cell ( IF 14.5 ) Pub Date : 2024-06-24 , DOI: 10.1016/j.molcel.2024.05.024
Mei-Sheng Xiao ₁ , Arun Prasath Damodaran ₁ , Bandana Kumari ₁ , Ethan Dickson ₁ , Kun Xing ₁ , Tyler A On ₂ , Nikhil Parab ₁ , Helen E King ₃ , Alexendar R Perez ₄ , Wilfried M Guiblet ₁ , Gerard Duncan ₅ , Anney Che ₆ , Raj Chari ₇ , Thorkell Andresson ₅ , Joana A Vidigal ₈ , Robert J Weatheritt ₉ , Michael Aregger ₂ , Thomas Gonatopoulos-Pournatzis ₁

Affiliation

RNA Biology Laboratory, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA.
Molecular Targets Program, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA.
EMBL Australia and Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA; Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, CA 94143, USA.
Protein Characterization Laboratory, Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21701, USA.
Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21701, USA.
Genome Modification Core, Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21702, USA.
Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
EMBL Australia and Garvan Institute of Medical Research, Sydney, NSW 2010, Australia; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2010, Australia.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

中文翻译：

基因组规模的外显子扰动筛选揭示了对细胞健康至关重要的外显子

CRISPR-Cas 技术已经改变了功能基因组学，但对单个外显子如何差异塑造细胞表型的了解仍然有限。在这里，我们在人类细胞系中优化并进行了大规模并行的外显子缺失和剪接位点突变筛选，以识别调节细胞适应性的外显子。促进健康的外显子在必需和高表达的基因中普遍存在，并且通常与蛋白质结构域和相互作用界面重叠。相反，适应度抑制外显子富含非必需基因，表现出较低的包含水平，并与本质上无序的区域和疾病相关突变重叠。对屏幕上的替代外显子 8 进行深入的机制研究表明，它的包含是 TFIID 通用转录起始复合物组装所必需的，从而调节全局基因表达输出。总的来说，我们的正交外显子扰动筛选建立了表型重要外显子的综合存储库，并揭示了控制细胞适应性和基因表达的调控机制。

更新日期：2024-06-24

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