A high-throughput methods SNIPR-Nm reveals small RNA methylation ratio

Tang, Y.; Li, Y.; Zhu, L.; Chi, X.; Wu, Y.; Liu, K.; Tang, H.; Liu, T.; Hu, Y.; Zhao, G.; Chen, J.-Q.; Chen, Q.

doi:10.64898/2025.12.11.693694

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bioRxiv · 10.64898/2025.12.11.693694

A high-throughput methods SNIPR-Nm reveals small RNA methylation ratio

Tang, Y.·Li, Y.·Zhu, L.·Chi, X.·Wu, Y.·Liu, K.·Tang, H.·Liu, T.·Hu, Y.·Zhao, G.·Chen, J.-Q.·Chen, Q.

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Abstract

Small RNAs regulate gene expression and genome stability, and their functions are shaped by 3'-terminal 2'-O-methylation (Nm). Although Nm is universal in plants and mainly marks animal piRNAs, recent evidence reveals Nm on mammalian miRNAs, highlighting broader regulatory roles. However, current sequencing underestimates Nm-modified RNAs due to ligation bias, and existing qPCR-based methods cannot simultaneously quantify abundance and modification, leaving Nm dynamics largely unexplored. We developed a unified enzymatic strategy that integrates poly(A) polymerase and a thermostable ligase to measure both small RNA abundance and 3'-end Nm status. Applied to mouse testis, it enables robust quantification of Nm ratios for piRNAs and miRNAs. Our method offers a versatile tool for dissecting small RNA methylation in development and disease.

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Tang, Y., Li, Y., Zhu, L., Chi, X., Wu, Y., Liu, K., Tang, H., Liu, T., Hu, Y., Zhao, G., Chen, J.-Q., Chen, Q.. 2025-12-13. A high-throughput methods SNIPR-Nm reveals small RNA methylation ratio. https://doi.org/10.64898/2025.12.11.693694

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