CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

High-throughput sequencing reveals the complex landscape of small non-coding RNAs (sRNAs). However, it is limited by requiring 5'-monophosphate and 3'-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkBD135S treatment reveals methylation nucleosides on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs across mouse tissues. We also observed the transition of sRNomes during hepatic reprogramming. Knock down of mesenchymal-stem-cell-enriched U1-5' snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.

高通量测序揭示了小分子非编码RNA（small non-coding RNAs，sRNAs）的复杂特征图谱。然而，该技术存在两大局限：一是要求RNA分子具备5'-单磷酸与3'-羟基基团以完成接头连接，二是会被干扰逆转录过程的甲基化核苷所阻碍。本研究开发了基于Cap-Clip酸性焦磷酸酶（Cap-Clip acid pyrophosphatase）、T4多核苷酸激酶（T4 polynucleotide kinase，PNK）以及AlkB/AlkB(D135S)的小分子非编码RNA测序技术（CPA-seq），用以检测并定量带有末端多态性与核苷甲基化修饰的sRNAs。CPA-seq鉴定出了大量此前未被检测到的sRNAs。对比经与未经AlkB/AlkB(D135S)处理的sRNAs，可揭示sRNAs上的甲基化核苷修饰。借助CPA-seq，我们解析了9种小鼠组织的sRNA转录组（sRNomes），并报道了小鼠不同组织间sRNAs广泛的组织特异性表达差异。我们还观察到肝脏重编程过程中sRNomes的动态变化。敲低富集于间充质干细胞的U1-5'小核RNA（U1-5' snsRNA）可促进肝脏重编程。CPA-seq是一款兼具高灵敏度与高特异性的高效工具，可用于分析带有甲基化核苷修饰与多样化末端结构的sRNAs。