Genome-wide base editor screen identifies regulators of protein abundance in yeast.
Genome-wide base editor screen identifies regulators of protein abundance in yeast.
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DOI：
10.7554/elife.79525
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发表时间：
2022-11-03
期刊：
影响因子：
7.7
通讯作者：
Kruglyak L
中科院分区：
文献类型：
--
作者：
Schubert OT;Bloom JS;Sadhu MJ;Kruglyak L
蛋白质是细胞中的关键分子，它们的丰度不仅在基因表达水平上而且在转录后水平上受到广泛调节。在这里，我们描述了在酵母中的遗传筛选，使蛋白质丰度调控如何在基因组中编码的系统表征。该筛选结合了CRISPR/Cas9碱基编辑器以引入点突变，并对内源性蛋白质进行荧光标记，以促进流式细胞术读数。我们首先用单个gRNA以及在阳性和阴性选择筛选中对酵母中的碱基编辑器性能进行基准测试。然后，我们研究了16，452种遗传扰动对代表各种细胞功能的11种蛋白质丰度的影响。我们发现了数百种调控关系，包括GAPDH同工酶Tdh 1/2/3和Ras/PKA通路之间的新联系。许多已鉴定的调节因子对11种蛋白质中的一种具有特异性，但我们也发现了一些基因，这些基因在受到干扰时会影响大多数测试蛋白质的丰度。虽然更具体的调节剂通常在转录上起作用，但广泛的调节剂通常在蛋白质翻译中起作用。总的来说，我们的新筛选方法为蛋白质调控网络的组件，规模和连通性提供了前所未有的见解。
Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.