奥地利维也纳生物中心Stefan L. Ameres、Alexander Stark、Ulrich Elling和Julius Brennecke小组取得一项新突破。他们在哺乳动物细胞中利用ORFtag实现了蛋白质组的规模化标记和功能筛选。相关论文于2024年7月5日发表在《自然-方法学》杂志上。

据介绍，系统地确定蛋白质功能是现代生物学的一个重要目标，但目前的方法难以实现该目的。

研究人员建立了ORFtag技术，这是一种多功能、低成本、高效率的方法，可在蛋白质组规模上对蛋白质进行大规模并行标记和功能检测。ORFtag使用带有启动子、肽标签和剪接供体的逆转录病毒载体，在标签和内源性开放阅读框（ORF）之间建立融合。

研究通过对小鼠胚胎干细胞中的转录激活因子、抑制因子和转录后调节因子进行功能筛选，证明了ORFtag 的实用性。每次筛选都能检测出已知的调控因子并发现新的调控因子，包括其他方法无法进行的长ORF。研究还发现，Zfp574是一种高度选择性的转录激活因子，并且致癌融合体通常具有转录激活因子的功能。

附：英文原文

Title: Proteome-scale tagging and functional screening in mammalian cells by ORFtag

Author: Nemko, Filip, Himmelsbach, Moritz, Loubiere, Vincent, Yelagandula, Ramesh, Pagani, Michaela, Fasching, Nina, Brennecke, Julius, Elling, Ulrich, Stark, Alexander, Ameres, Stefan L.

Issue&Volume: 2024-07-05

Abstract: The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

DOI: 10.1038/s41592-024-02339-x

Source: https://www.nature.com/articles/s41592-024-02339-x