Russo Joseph, Heck Adam M, Wilusz Jeffrey, Wilusz Carol J
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States.
Program in Cell & Molecular Biology, Colorado State University, Fort Collins, CO 80525, United States.
Methods. 2017 May 1;120:39-48. doi: 10.1016/j.ymeth.2017.02.003. Epub 2017 Feb 20.
Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates. This approach has been widely adopted for measuring mRNA half-lives on a global scale, but has proven challenging to use for analysis of single genes. We present a series of normalization and quality assurance steps to be used in combination with 4-thiouridine pulse labeling of cultured eukaryotic cells. Importantly, we demonstrate how the relative amount of 4sU-labeled nascent RNA influences accurate quantification. The approach described facilitates reproducible measurement of individual mRNA half-lives using 4-thiouridine and could be adapted for use with other nucleoside analogs.
mRNA 衰变率的变化与转录变化密切协调，在发育和疾病过程中，这些事件共同对基因表达产生深远影响。评估 mRNA 衰变的传统方法依赖于转录抑制，而这可能会改变 mRNA 衰变率并混淆解释。最近，代谢标记与化学修饰以及标记 RNA 的分级分离相结合，使得新生转录本得以分离，并随后计算出 mRNA 衰变率。这种方法已被广泛用于在全球范围内测量 mRNA 半衰期，但事实证明，将其用于单个基因的分析具有挑战性。我们提出了一系列标准化和质量保证步骤，以便与培养的真核细胞的 4-硫尿苷脉冲标记结合使用。重要的是，我们展示了 4sU 标记的新生 RNA 的相对量如何影响准确定量。所描述的方法有助于使用 4-硫尿苷对单个 mRNA 半衰期进行可重复测量，并且可以适用于与其他核苷类似物一起使用。
