Whalen Courtney, Tuohy Christine, Tallo Thomas, Kaufman James W, Moore Claire, Kuehner Jason N
Department of Nutritional Sciences, College of Health and Human Development, The Pennsylvania State University, University Park, PA 16802.
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605.
G3 (Bethesda). 2018 May 31;8(6):2043-2058. doi: 10.1534/g3.118.200072.
Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast , Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DNA repair gene, which includes a portion of the 5'-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 -acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating expression, which can be modulated during the DNA damage response.
RNA聚合酶II（Pol II）活性的终止通过分离普遍存在的转录单元并影响RNA的命运和功能，在细胞中发挥着至关重要的作用。在酵母中，Pol II的终止由切割和聚腺苷酸化因子（CPF-CF）以及Nrd1-Nab3-Sen1（NNS）复合物执行，它们分别主要作用于mRNA和非编码RNA基因。Pol II过早终止（衰减）有助于基因调控，但其发生率和生物学意义的相关知识有限。特别是，目前尚不清楚CPF-CF和NNS复合物之间会发生多少相互作用，以及在应激适应过程中Pol II衰减是如何被调节的。在本研究中，我们在DNA修复基因中鉴定出一个衰减子，它包括一部分5'非翻译区（UTR）和上游开放阅读框（ORF）。利用基于质粒的报告基因系统，我们对14个终止突变体及其赋予Pol II通读缺陷的能力进行了遗传筛选。该衰减子表现为一种混合终止子，严重依赖CPF-CF和Sen1，但不涉及Nrd1和Nab3。我们的遗传筛选鉴定出22个起作用的点突变，这些突变聚集在四个区域，包括一个聚腺苷酸化位点效率元件，它与其同源结合蛋白Hrp1发生遗传相互作用。在报告基因背景之外，一个衰减子突变体增加了mRNA和蛋白质表达，加剧了组成型活性Def1蛋白的毒性。总体而言，我们的数据支持转录衰减在调节基因表达中具有生物学意义，并且在DNA损伤反应过程中可以被调节。
