Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, Colorado 80045, USA
RNA. 2023 Jun;29(6):847-861. doi: 10.1261/rna.079290.122. Epub 2023 Feb 28.
Ligation by plant and fungal RNA ligases yields an internal 2'-phosphate group on each RNA ligation product. In budding yeast, this covalent mark occurs at the splice junction of two targets of ligation: intron-containing tRNAs and the messenger RNA The repertoire of RNA molecules repaired by RNA ligation has not been explored due to a lack of unbiased approaches for identifying RNA ligation products. Here, we define several unique signals produced by 2'-phosphorylated RNAs during nanopore sequencing. A 2'-phosphate at the splice junction of mRNA inhibits 5' → 3' degradation, enabling detection of decay intermediates in yeast RNA repair mutants by nanopore sequencing. During direct RNA sequencing, intact 2'-phosphorylated RNAs on and tRNAs produce diagnostic changes in nanopore current properties and base calling features, including stalls produced as the modified RNA translocates through the nanopore motor protein. These approaches enable directed studies to identify novel RNA repair events in other contexts.
植物和真菌 RNA 连接酶的连接在每个 RNA 连接产物上产生内部 2'-磷酸基团。在酿酒酵母中，这种共价标记发生在两个连接靶标（内含子 tRNA 和信使 RNA）的剪接连接处。由于缺乏用于鉴定 RNA 连接产物的无偏方法，因此尚未探索通过 RNA 连接修复的 RNA 分子的库。在这里，我们在纳米孔测序过程中定义了几种由 2'-磷酸化 RNA 产生的独特信号。mRNA 剪接连接处的 2'-磷酸基团抑制 5'→3'降解，从而使纳米孔测序能够检测酵母 RNA 修复突变体中的降解中间产物。在直接 RNA 测序过程中，完整的 2'-磷酸化 RNA 在 mRNA 和 tRNA 上产生纳米孔电流特性和碱基调用特征的诊断性变化，包括作为修饰的 RNA 通过纳米孔马达蛋白转运而产生的停顿。这些方法可用于定向研究以在其他情况下鉴定新的 RNA 修复事件。
