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.

中文翻译：

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使用高通量测序确定降解中间体和组蛋白 mRNA 的 3' 到 5' 降解途径

mRNA 的半衰期是影响 mRNA 稳态水平的重要参数。mRNA 水平的快速变化可能是由于 mRNA 的半衰期缩短所致。为特定类别的 mRNA 建立 mRNA 降解的详细途径需要能够分离 mRNA 降解中间体。高通量测序提供了一种检测这些中间体的方法。在这里，我们描述了一种确定 3' 到 5' 降解中间体的方法。表征这些中间体不仅需要确定分子的精确 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非模板核苷酸。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更改。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。这种方法可能适用于任何 RNA，我们讨论了将方法扩展到包括同时测定同一 RNA 分子的 3' 和 5' 端的可能策略。