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. 2007 Sep;13(9):1397-401.
doi: 10.1261/rna.659307. Epub 2007 Jul 24.

The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs

Yohei Kirino, Zissimos Mourelatos

The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs

Yohei Kirino et al. RNA. 2007 Sep.

Abstract

Piwi-interacting RNAs (piRNAs) are a novel class of small regulatory RNAs that are expressed specifically and abundantly in germ cells. Mammalian piRNAs are 26-31 nucleotides in length and bind to Piwi proteins, but their function and biogenesis remain elusive. We previously showed that mammalian piRNAs are 2'-O-methylated at their 3' termini. The biosynthetic mechanism and function of this modification is unknown. Here, we report that the mouse homolog (mHEN1) of HEN1, a plant microRNA (miRNA) 2'-O-methyltransferase, is expressed specifically in testis and methylates 3' termini of piRNAs in vitro. These findings provide insight into the biogenesis of piRNAs.

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Figures

FIGURE 1.
FIGURE 1.
The mRNA of mouse HEN1 (mHEN1) is expressed specifically in testis. Analysis of mHEN1 expression in mouse tissues. EF-1β was used as a control of a ubiquitously expressed mRNA.
FIGURE 2.
FIGURE 2.
mHEN1 has piRNA methyltransferase activity in vitro. (A) A diagram of mHEN1 protein showing the putative methyltransferase domain. The sequences of the domain (54–58 amino acids) are mutated in mHEN1-m as indicated. (B) Coomassie-blue stained SDS-PAGE of the affinity purified GST-mHEN1 and GST-mHEN1-m proteins. The full-length mHEN1 proteins are indicated by the arrowhead. (C) The RNA substrates (shown in Table 1) were incubated with mHEN1 protein and [14C] SAM. [14C]-labeled RNA was resolved by UREA-PAGE and detected by autoradiography. (D) 2D-TLC analysis of in vitro methylated nucleotides derived from piR-3 (28G) and piR-3 (28C). The positions of 5′monophosphonucleotides, pA, pG, pC, and pU, detected by UV shadowing are indicated. Solvent dimensions 1 and 2 are detailed in Materials and Methods.
FIGURE 3.
FIGURE 3.
In vitro mHEN1 methylation of various RNA substrates (A) Methylation of indicated RNA substrates was detected by liquid scintillation counting. The amount of input RNA was defined as 100. The averages of five independent experiments with SD values are shown. (B) Methylation of RNA substrates was analyzed by UREA-PAGE and autoradiography.
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