RNA. 2014 May;20(5):732-45. doi: 10.1261/rna.042663.113. Epub 2014 Mar 26.
It has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter using multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 nonessential yeast genes revealed striking phenotypic overlaps between chromatin remodeling, histone modification, and pre-mRNA splicing. Additionally, we developed a copper-inducible reporter, with which we demonstrate that 5-fluorouracil mimics the mRNA decay phenotype of cells lacking the 3'-5' exonuclease Rrp6p. Our reporter is capable of performing high-throughput, rapid, and large-scale screens to identify and characterize genetic and chemical perturbations of the major eukaryotic gene expression processes.
越来越明显的是，真核生物中的基因表达过程涉及许多复杂的、独立的大分子机器之间的通信和协调。为了查询这些过程，并在芽殖酵母酿酒酵母中探索它们之间的潜在关系，我们设计了一种使用多色高通量流式细胞术的通用报告器。由于其设计，这种单一的报告器对于许多基因表达缺陷，包括转录、组蛋白修饰、前体 mRNA 剪接、mRNA 输出、无意义介导的衰变和 mRNA 降解，都表现出独特的特征。在 4967 个非必需酵母基因中的报告器分析显示，染色质重塑、组蛋白修饰和前体 mRNA 剪接之间存在显著的表型重叠。此外，我们开发了一种铜诱导的报告器，用它我们证明了 5-氟尿嘧啶模拟了缺乏 3'-5'外切酶 Rrp6p 的细胞的 mRNA 衰变表型。我们的报告器能够进行高通量、快速和大规模筛选，以识别和表征主要的真核生物基因表达过程中的遗传和化学干扰。
