Unbiased screen of RNA tailing enzymes reveals a poly(UG) polymerase required for genome integrity and RNA silencing

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.核糖核苷酸转移酶（ribonucleotidyl transferases，rNTases）能够将非模板依赖型核糖核苷酸添加至各类RNA分子中。我们在酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）中开发了一种筛选策略，可实现以单核苷酸分辨率鉴定不同来源的候选酶所添加的RNA序列。本研究测定了19种此前未被表征的酶的核糖核苷酸转移酶活性。除了可添加poly(A)与poly(U)的酶之外，我们还鉴定出一种加C的酶，该酶大概率参与真核生物中以双酶系统向转运RNA（transfer RNA，tRNA）添加CCA序列的过程；同时鉴定出一种对核苷酸无明显偏好性、可向RNA添加核苷酸的核苷酸转移酶；以及一种聚(UG)聚合酶——秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）MUT-2，该酶可交替添加尿苷与鸟苷以形成聚(UG)尾。已知MUT-2参与部分类型的RNA沉默过程，而该酶中导致沉默功能缺陷的突变体，在本研究的实验体系中同样无法形成聚(UG)尾。我们推测，MUT-2的聚(UG)聚合酶活性对于维持基因组完整性以及介导RNA沉默至关重要。