Department of Orthopaedics and Rehabilitation, Penn State University, College of Medicine, Hershey, PA 17033-0850, USA; Department of Biochemistry and Molecular Biology, Penn State University, College of Medicine, Hershey, PA 17033-0850, USA.
Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA.
Methods. 2019 Jan 1;152:18-22. doi: 10.1016/j.ymeth.2018.05.010. Epub 2018 May 17.
MicroRNAs (miRNAs) comprise a class of small non-coding RNAs that regulate the stability and/or translatability of most protein-coding transcripts. Steady-state levels of mature miRNAs can be controlled through mechanisms that influence their biogenesis and/or decay rates. Pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway that promotes the sequence-specific endonucleolytic decay of miRNAs that harbor a CA and/or UA dinucleotide. Here, we describe an in vitro assay for evaluating the susceptibility of AGO2-loaded miRNAs to degradation by different classes of nucleases. This in vitro approach can be used to complement in vivo studies that aim to identify novel miRNA decay factors.
MicroRNAs (miRNAs) 是一类小的非编码 RNA，可调节大多数蛋白质编码转录物的稳定性和/或翻译能力。成熟 miRNA 的稳态水平可以通过影响其生物发生和/或降解率的机制来控制。介导成熟 miRNA 降解的途径比介导 miRNA 生物发生的途径理解得更少。我们最近描述了 Tudor-葡萄球菌/微球菌样核酸内切酶（TSN）介导的 miRNA 降解（TumiD）作为一种细胞途径，可促进含有 CA 和/或 UA 二核苷酸的 miRNA 的序列特异性内切核酸酶降解。在这里，我们描述了一种评估 AGO2 加载的 miRNA 对不同类核酶降解敏感性的体外测定法。这种体外方法可用于补充旨在鉴定新的 miRNA 降解因子的体内研究。
