RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.
RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.
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DOI：
10.1534/g3.118.200072
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发表时间：
2018-05-31
期刊：
影响因子：
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通讯作者：
Kuehner JN
中科院分区：
文献类型：
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作者：
Whalen C;Tuohy C;Tallo T;Kaufman JW;Moore C;Kuehner JN
RNA聚合酶II（Pol II）活性的终止通过分离普遍存在的转录单位并影响RNA命运和功能而发挥重要的细胞作用。在酿酒酵母中，Pol II终止是通过切割和多聚腺苷酸化因子（CPF-CF）和Nrd 1-Nab 3-Sen 1（NNS）复合物进行的，其主要分别在mRNA和非编码RNA基因处起作用。提前Pol II终止（衰减）有助于基因调控，但其患病率和生物学意义的知识有限。特别是，目前还不清楚有多少串扰之间发生CPF-CF和NNS复合物，以及如何Pol II衰减调制过程中的压力适应。在本研究中，我们在DEF 1 DNA修复基因中鉴定了一个衰减子，它包括一部分5′-非翻译区（UTR）和上游开放阅读框（ORF）。使用质粒为基础的报告基因系统，我们进行了遗传筛选的14个终止突变体和他们的能力，赋予Pol II通读缺陷。DEF 1衰减子表现为混合终止子，严重依赖于CPF-CF和Sen 1，但没有Nrd 1和Nab 3参与。我们的遗传选择确定了22个顺式作用点突变，聚类成四个区域，包括多聚腺苷酸化位点效率元件，与其同源结合蛋白Hrp 1的遗传相互作用。在报告基因背景之外，DEF 1衰减子突变体增加mRNA和蛋白质表达，加剧了组成型活性Def 1蛋白的毒性。总的来说，我们的数据支持转录衰减在调节DEF 1表达中的生物学重要作用，其可以在DNA损伤反应期间进行调节。
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 Saccharomyces cerevisiae, 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 DEF1 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 DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-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 DEF1 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 DEF1 expression, which can be modulated during the DNA damage response.