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

High-throughput sequencing reveals the complex landscape of small noncoding 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/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs.


Fig: a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m7G and m3G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m1G, m3C, and m1A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m7G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m1G, m3C, and m1A of small RNAs extracted from HEK293T cells (n = 3).

We also observed the transition of sRNomes during hepatic reprogramming. Knockdown 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.

Wang, H., Huang, R., Li, L. et al. CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini. Cell Discov 7, 25 (2021).

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