High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq

Measurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from mature tRNA with a simple-to-use computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in multiple eukaryotes and is applicable to any organism with a known genome. Development of method for high-throughput sequencing, quantitation and data analysis of tRNA pools in multiple eukaryotic species. Evidence of application for assesing differential expression of tRNAs between two human cell lines, investigating tRNA charging fraction, and extensive modification analysis.

细胞转运RNA（tRNA）丰度的检测长期面临两大技术瓶颈：一是修饰核苷会普遍阻断互补脱氧核糖核酸（cDNA）的合成过程，二是tRNA基因之间存在高度序列同源性。本研究开发的修饰诱导错配tRNA测序（modification-induced misincorporation tRNA sequencing, mim-tRNAseq）突破了上述局限，该方法整合了从成熟tRNA构建全长cDNA文库的实验流程与一套操作简便的计算分析工具包。本方法可在多种真核生物中精准捕获tRNA丰度与修饰状态信息，且适用于任何具有已知基因组的生物体。该方法实现了多种真核生物tRNA群体的高通量测序、定量及数据分析流程，并已被证实可应用于多项研究场景：包括评估两种人类细胞系间tRNA的差异表达水平、探究tRNA氨酰化比例，以及开展大规模修饰状态分析。