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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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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