Marzi Matteo J, Nicassio Francesco
Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan, Italy.
Methods Mol Biol. 2018;1823:141-152. doi: 10.1007/978-1-4939-8624-8_11.
MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (<18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates.
微小RNA（miRNA）是一类在进化上保守的短链单链非编码RNA（长度<18 - 22个核苷酸），在高等真核生物中参与基因表达的转录后调控。miRNA的丰度是控制其活性的关键特征，因此，许多机制在转录和转录后水平精细调节miRNA水平。包括我们的研究在内的最新证据突出了miRNA降解作为控制miRNA库的一种机制的作用。在本章中，我们描述了一种优化的方法来确定哺乳动物细胞中miRNA的降解速率。我们的方法基于用4 - 硫尿苷（4sU）进行代谢脉冲标记，4sU是一种尿苷类似物，可掺入新生RNA中，并允许进行硫醇特异性生物素化以及对标记RNA进行选择性下拉。特别是，鉴于miRNA的平均半衰期长且生物发生过程复杂，我们开发了一种“脉冲追踪”方案，即在长时间标记期（2 - 3小时脉冲）后从培养基中去除4sU，并在不同时间点纯化标记RNA以测量标记分子的降解。通过将基于4sU的“脉冲追踪”方法与高通量小RNA测序（sRNAseq）相结合，有可能在全基因组水平上量化miRNA的降解速率。
