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Pimet, the Drosophila Homolog of HEN1, Mediates 2 -O-Methylation of Piwi- Interacting Rnas at Their 3 Ends

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Pimet, the Drosophila Homolog of HEN1, Mediates 2 -O-Methylation of Piwi- Interacting Rnas at Their 3 Ends Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION the AGO subfamily function in the microRNA (miRNA) Pimet, the Drosophila pathway and RNA interference (RNAi) by binding with homolog of HEN1, mediates miRNAs and short interfering RNAs (siRNAs), respec- tively (Okamura et al. 2004; Tomari et al. 2004; Miyoshi -2؅-O-methylation of Piwi- et al. 2005). Recent studies have revealed that PIWI pro interacting RNAs at their teins are specifically associated with a subset of endog- enous small RNAs, termed Piwi-interacting RNAs 3؅ ends (piRNAs; in flies, these are also called repeat-associated siRNAs, or rasiRNAs), which are distinct from miRNAs Kuniaki Saito,1 Yuriko Sakaguchi,2 in many respects such as size, expression pattern, and Takeo Suzuki,2 Tsutomu Suzuki,2 even function (O’Donnell and Boeke 2007). Unlike Haruhiko Siomi,1 and Mikiko C. Siomi1,3 miRNAs, piRNAs are 24–30 nt long, are expressed only in gonads, and function in genome surveillance through 1Institute for Genome Research, University of Tokushima, association with the PIWI proteins (for review, see Kim Tokushima 770-8503, Japan; 2Department of Chemistry 2006; Saito et al. 2006; Vagin et al. 2006; Brennecke et al. and Biotechnology, Graduate School of Engineering, 2007; Gunawardane et al. 2007; Houwing et al. 2007) by The University of Tokyo, Tokyo 113-8656, Japan silencing transposable elements that have sufficient po- tency to invade the genome by inserting themselves into Piwi-interacting RNAs (piRNAs) consist of a germline- DNA elements. specific group of small RNAs derived from distinct in- Recently, a model for piRNA biogenesis in Drosophila tergenic loci in the genome. piRNAs function in silenc- was proposed (Brennecke et al. 2007; Gunawardane et al. ing selfish transposable elements through binding with 2007). In this model, PIWI proteins determine and form Ј the PIWI subfamily proteins of Argonautes. Here we 5 ends of piRNAs, which will then be loaded onto the other PIWI members, by cleaving their target transcripts show that piRNAs in Drosophila are 2؅-O-methylated at -؅ in a manner depending on the associated piRNA se their 3 ends. Loss of Pimet (piRNA methyltransferase), quence. This process not only continuously generates the Drosophila homolog of Arabidopsis HEN1 methyl- piRNAs but also keeps destroying target RNAs tran- transferase for microRNAs (miRNAs), results in loss of scribed from selfish genes. Fish and mammals may have .2؅-O-methylation of fly piRNAs. Recombinant Pimet similar mechanisms (Aravin et al. 2007; Houwing et al shows single-stranded small RNA methylation activity 2007). However, many aspects of piRNA biogenesis in in vitro and interacts with the PIWI proteins within Drosophila remain unclear; for example, protein factor(s) Pimet mutant ovary. These results show that Pimet me- determining and forming the 3Ј ends of piRNAs are not diates piRNA 2؅-O-methylation in Drosophila. identified. How nascent piRNAs are loaded onto the other PIWI members also has not been elucidated. Supplemental material is available at http://www.genesdev.org. In plants, miRNAs are methylated at the 2Ј-terminal hydroxyl group on the ribose at the last nucleotide (Yu et Received April 20, 2007; revised version accepted May 24, al. 2005). A factor responsible for such modification in 2007. Arabidopsis is HEN1 (Yu et al. 2005). This is a protein containing a double-stranded RNA (dsRNA)-binding do- Small, 20- to 30-nucleotide (nt)-long, RNAs trigger gene main at the N terminus and a catalytic domain at the C silencing mechanisms, collectively called RNA silenc- terminus that transfers methyl groups from S-adenosyl ing, which include cleavage-dependent and -independent methionine (SAM) to miRNA/miRNA* duplexes. Five mRNA decay, translational repression, DNA methyl- consecutive residues (Asp–Phe–Gly–Cys–Gly) residing ation, and heterochromatin formation in organisms in the catalytic domain of Arabidopsis HEN1 correspond ranging from fission yeast to humans (Tomari and to the SAM-binding motif. Mutations introduced in the Zamore 2005; Zaratiegui et al. 2007). RNA silencing- SAM-binding motif abolish the methylation activity. In triggering small RNAs are processed from long precur- the hen1 mutant, miRNA are reduced in abundance and sors and are then loaded onto Argonaute proteins (Parker when detectable are uridylated, a cause of miRNA de- and Barford 2006), guiding the Argonautes to specific se- stabilization (Li et al. 2005). It can be concluded that quences within the target RNAs (Tomari and Zamore HEN1 is the stabilizer of miRNAs in plants. Arabidopsis 2005; Pillai et al. 2007). HEN1 methylates even siRNA duplexes with perfect se- Numbers of the Argonaute protein family members, quence complementarity (Yang et al. 2006), but single- defined by the PAZ and PIWI domains, differ among spe- stranded small RNAs and pre-miRNAs forming a hairpin cies (Carmell et al. 2002); for instance, fly has five mem- structure fail to be methylated in vitro by recombinant bers (Williams and Rubin 2002) whereas mouse has HEN1 (Yu et al. 2005). It is thus postulated that miRNA seven. The Argonautes can be divided into two sub- methylation by HEN1 occurs between two steps during groups, the AGO and PIWI subfamilies, according to se- the miRNA maturation process; namely, after Dicer-me- quence similarity. In Drosophila, AGO1 and AGO2 of diated excision of miRNA/miRNA* duplexes from the precursors and before the unwinding process of the du- plexes. [Keywords: piRNA; 2Ј-O-methylation; HEN1; Pimet; Drosophila] Ј 3Corresponding author. piRNAs in Drosophila are modified at their 3 ends, as E-MAIL [email protected]; FAX 81 88 6339451. judged by their resistance to periodate oxidation/␤-elimi- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1563607. nation (Vagin et al. 2006; Gunawardane et al. 2007). In GENES & DEVELOPMENT 21:1603–1608 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 1603 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Saito et al. this study, we determined that piRNAs in Drosophila AGO1 increased mobility by nearly 2 nt (Fig. 1B), a fea- are 2Ј-O-methylated at their 3Ј ends, as in mouse and rat ture of RNAs bearing a 2Ј-, 3Ј-terminal-cis-diol group (Houwing et al. 2007; Kirino and Mourelatos 2007; (Hutvagner et al. 2001). This is consistent with the fact Ohara et al. 2007). In Drosophila, CG12367 encodes a that animal miRNAs are not modified at their 3Ј ends protein showing similarity at the peptide levels with the (Elbashir et al. 2001; Hutvagner et al. 2001). piRNAs ex- methyltransferase catalytic domain of Arabidopsis pressed in mouse and rat have recently been shown to be HEN1. Loss of CG12367 results in loss of piRNA meth- 2Ј-O-methylated at their 3Ј ends (Houwing et al. 2007; ylation in vivo; thus, we refer to this as Pimet (for piRNA Kirino and Mourelatos 2007; Ohara et al. 2007). To iden- methyltransferase). Indeed, Aub- and Piwi-associated tify the terminal modification of piRNA in Drosophila, piRNAs isolated from Pimet mutant ovary showed sen- Aub-associated piRNAs were gel-purified and digested sitivity to oxidation/␤-elimination as oppose to those in by RNase T2. The digest was analyzed by capillary liquid wild-type ovary. Recombinant Pimet showed activity in chromatography (LC) and mass spectrometry (MS) (Fig. vitro for transferring methyl groups from SAM to single- 2). In this treatment, only 3Ј-terminal residues are con- stranded small RNAs bearing a 2Ј,3Ј-terminal-cis-diol verted to nucleosides (Ohara et al. 2007). As shown in group, but not to 2Ј-O-methylated small RNAs. Aub- Figure 2B, four kinds of 2Ј-O-methyl nucleosides were associated piRNAs from Pimet mutant ovary were meth- clearly observed as major products. Each proton adduct ylated by recombinant Pimet in vitro; by contrast, was further analyzed by MS/MS using collision-induced AGO1-associated miRNAs did not serve as a substrate dissociation (CID). Selected reaction monitoring (SRM) for the methylation. Taken together, these results show for base-related product ions (Fig. 2C) and mass spectrum that Pimet is the factor responsible for piRNA methyl- (Supplementary Fig. S1) of each 2Ј-O-methyl nucleoside ation in Drosophila germlines. also confirmed that a methyl group is attached to each ribose portion. To eliminate the possibility that they were 3Ј-O-methyl nucleosides, RNase T2-digested Results and Discussion piRNAs were coinjected with a series of synthetic 3Ј-O- piRNAs in fly ovary are 2Ј-O-methylated methyl nucleosides. Each 3Ј-O-methyl nucleoside eluted Ј at their 3Ј ends at a distinct retention time from each 3 -terminal nucleoside of piRNAs (Fig. 2D). On the other hand, each piRNAs in fly ovary are resistant to periodate oxidation/ synthetic 2Ј-O-methyl nucleoside eluted at the same re- ␤-elimination treatment, indicating that these piRNAs tention time with its corresponding 3Ј-terminal nucleo- are modified at their 3Ј ends (Vagin et al. 2006; Gunawar- side of piRNAs (data not shown). These results indicate dane et al. 2007). In this study, similar experiments were that piRNAs in flies are almost entirely methylated at performed for piRNAs associated with Aub and Piwi in the 2Ј-hydroxyl group on the ribose of the last nucleo- ovary. In both cases piRNAs did not gain mobility even tide. after periodate oxidation/␤-elimination treatment (Fig. Ј 1A), indicating that they are modified at their 3 ends. CG12367 is involved in piRNA methylation Under the same conditions, miRNAs associated with in the ovary In Arabidopsis, miRNAs are methylated at their 3Ј ends by HEN1 while still in a duplex form with miRNA* molecules (Yu et al. 2005). HEN1 is a methyltransferase that contains two characteristic motifs, a dsRNA-bind- ing domain and a catalytic domain necessary for the methyltransferase activity.
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