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Updated: Feb 11, 2026
Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC
Michael D Horwich1, Chengjian Li, Christian Matranga
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Abstract:
Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways [1, 2]. In the RNA interference (RNAi) pathway [3], small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression [4]. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons [5-9]. Here, we report that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs [10] and mouse and rat piRNAs [11, 12, 13]. Plant small-RNA methylation is catalyzed by the protein HEN1 [10, 14, 15]. We find that DmHen1, the Drosophila homolog of HEN1, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs-which derive from double-stranded precursors-and piRNAs-which do not [8, 13]. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after an Argonaute-bound siRNA duplex is converted to single-stranded RNA.
Insights
Drosophila melanogaster's DmHen1 protein methylates small silencing RNAs, including piRNAs (Piwi-interacting RNAs) and siRNAs (small interfering RNAs). This 2'-O-methylation is crucial for maintaining piRNA length, abundance, and function in gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- RNA Biology
Background:
- Small silencing RNAs, including siRNAs, miRNAs, and piRNAs, regulate gene expression through RNA-silencing pathways.
- piRNAs are known to defend the genome against transposons.
- A 2 -O-methyl modification on the 3 termini of small RNAs has been observed in plants and mammals.
Purpose of the Study:
- To investigate the role of the Drosophila HEN1 homolog (DmHen1) in small RNA modification.
- To determine if DmHen1 methylates piRNAs and siRNAs in Drosophila.
- To understand the functional consequences of DmHen1-mediated methylation on piRNA and siRNA pathways.
Main Methods:
- Identification and characterization of DmHen1 in Drosophila.
- Biochemical assays to test DmHen1 activity on siRNAs and piRNAs.
- Analysis of small RNA populations in DmHen1 knockout or mutant flies.
Main Results:
- Drosophila piRNAs possess a 2 -O-methyl group at their 3 termini.
- DmHen1 catalyzes the 2 -O-methylation of both siRNAs and piRNAs.
- Loss of DmHen1 function leads to reduced piRNA length and abundance, and impaired piRNA function.
- DmHen1 acts on single-stranded RNAs, accommodating both siRNA and piRNA substrates.
Conclusions:
- DmHen1 is essential for the 2 -O-methylation of piRNAs and siRNAs in Drosophila.
- This modification is critical for maintaining the stability and function of piRNAs.
- DmHen1's single-strand activity explains its role in methylating both duplex-derived siRNAs and non-duplex piRNAs.
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