Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.

中文翻译：

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在核苷酸分辨率下解开 3'-末端 RNA 尿苷化

RNA 的转录后修饰，即所谓的“外转录组”，可以调节 RNA 的结构、稳定性、定位和功能。在几乎所有类别的 RNA 中都发现了许多修饰，包括信使 RNA (mRNA)、转移 RNA (tRNA)、核糖体 RNA (rRNA)、微小 RNA (miRNA) 和其他非编码 RNA (ncRNA)。这些修饰可能发生在内部（通过碱基或糖修饰），包括不同核苷酸位置的 RNA 甲基化，或通过末端核苷酸转移酶家族在某些转录物的 3' 端添加各种核苷酸。开发专门和准确地检测和映射这些修饰的方法对于理解单个 RNA 修饰的分子功能以及识别和表征可以读取、写入或擦除它们的蛋白质至关重要。在这里，我们专注于在最近发现的 Dis3l2 介导的 3' 末端尿苷基转移酶 (TUTases) (TUT4/7，也称为 Zcchc11/6) 和 3'-5' 外切糖核酸酶 Dis3l2 靶向的 RNA 种类的表征衰变 (DMD) 途径 - ncRNA 子集的专用质量控制途径。我们描述了用于在核苷酸级别分辨率下精确识别 3' 端修饰的详细方法，特别关注剪接体的 U1 和 U2 小核 RNA (snRNA) 组件。