Small noncoding RNAs of 20–30 nucleotides in length are key mediators of gene silencing. 2′-O-Methylation on the 3′ terminal nucleotide of several types of small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants, PIWI-interacting RNAs (piRNAs) in animals, and some siRNAs in Drosophila and Caenorhabditis elegans, provides a key protective mechanism against 3′ tailing- and trimming-mediated destabilization. The methylation reaction is catalyzed by the small RNA methyltransferase HUA ENHANCER 1 (HEN1). In this chapter, we describe a detailed protocol for analyzing 3′ end methylation status of plant miRNAs, which can be applicable to other types of small RNAs as well.
MicroRNA HEN1 Methylation β-elimination Denaturing PAGE Northern blot
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Work in the Ren laboratory is supported by grants from the National Key R&D Program of China (2016YFA0503200) and the National Natural Science Foundation of China (91740101, 31622009 and 31471221).
No conflict of interest was declared.
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