Department of Chemical Biology and Medicinal Chemistry, University of North Carolina, Chapel Hill, NC 27599, United States.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, United States.
Methods. 2019 Feb 15;155:104-115. doi: 10.1016/j.ymeth.2018.11.001. Epub 2018 Nov 5.
The half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3' to 5' degradation. Characterizing these intermediates requires not only determining the precise 3' end of the molecule to a single nucleotide resolution, but also the ability to detect and characterize any untemplated nucleotides present on the intermediates. We achieve this by ligating a known sequence to all the 3' termini in the cell, and then sequence the 3' termini and the ligated linker to identify any alterations to the genomic reference sequence. We have applied this method to characterize the intermediates in histone mRNA metabolism, allowing us to deduce the pathway of 3' to 5' degradation. This method can potentially be applied to any RNA, and we discuss possible strategies for extending the method to include simultaneous determination of the 3' and 5' end of the same RNA molecule.
mRNA 的半衰期是影响 mRNA 稳态水平的一个重要参数。mRNA 水平的快速变化可能是由于 mRNA 半衰期的缩短造成的。要确定特定类别 mRNA 的 mRNA 降解的详细途径，需要能够分离 mRNA 降解中间体。高通量测序为检测这些中间体提供了一种方法。在这里，我们描述了一种确定 3' 到 5' 降解过程中中间体的方法。对这些中间体进行特征分析不仅需要精确确定分子的 3' 末端到单个核苷酸分辨率，还需要能够检测和表征中间体上存在的任何无模板核苷酸。我们通过将已知序列连接到细胞中的所有 3' 末端来实现这一点，然后对 3' 末端和连接的接头进行测序，以确定对基因组参考序列的任何改变。我们已经应用这种方法来表征组蛋白 mRNA 代谢中的中间体，从而推断出 3' 到 5' 降解的途径。该方法可潜在地应用于任何 RNA，我们还讨论了将该方法扩展到同时确定同一 RNA 分子的 3' 和 5' 末端的可能策略。
