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, 21 (13), 1603-8

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

Kuniaki Saito et al. Genes Dev.

Abstract

Piwi-interacting RNAs (piRNAs) consist of a germline-specific group of small RNAs derived from distinct intergenic loci in the genome. piRNAs function in silencing selfish transposable elements through binding with the PIWI subfamily proteins of Argonautes. Here we show that piRNAs in Drosophila are 2'-O-methylated at their 3' ends. Loss of Pimet (piRNA methyltransferase), the Drosophila homolog of Arabidopsis HEN1 methyltransferase for microRNAs (miRNAs), results in loss of 2'-O-methylation of fly piRNAs. Recombinant Pimet shows single-stranded small RNA methylation activity in vitro and interacts with the PIWI proteins within Pimet mutant ovary. These results show that Pimet mediates piRNA 2'-O-methylation in Drosophila.

Figures

Figure 1.
Figure 1.
piRNAs associated with Aub and Piwi in fly ovary are methylated at their 3′ ends. (A) piRNA associated with Aub and Piwi in fly ovary were subjected to periodate oxidation/β-elimination chemical reactions. In both cases, piRNAs did not gain mobility, indicating that these piRNAs with Aub and Piwi were modified at their 3′ ends. Under these conditions, a synthetic 21-nt small RNA (luc guide siRNA) (Okamura et al. 2004) gained mobility by nearly 2 nt, a feature of RNAs bearing a 2′-, 3′-terminal-cis-diol group at the last nucleotide (Hutvagner et al. 2001). (B) miRNAs associated with AGO1 in fly ovary show that they are not modified at their 3′ ends, in contrast to plant miRNAs (Yu et al. 2005).
Figure 2.
Figure 2.
piRNAs associated with Aub in fly ovary are 2′-O-methylated at their 3′ ends. (A) Total ion chromatogram (TIC) of the piRNAs digested by RNase T2. The retention times of the observed 3′-phospho nucleotides (Cp, Up, Gp, and Ap) are indicated. The expected retention times of unmodified nucleosides (C, U, G, and A) are shown in gray. The considerable amount of adenosine observed is due to contamination (data not shown), as in the case of mouse piRNA MS analysis (Ohara et al. 2007). (B) Mass chromatograms for proton adducts of piRNA 3′-terminal nucleosides, 2′-O-Me C (m/z 258; black line), 2′-O-Me U (m/z 259; green line), 2′-O-Me G (m/z 298; red line), and 2′-O-Me A (m/z 282; blue line). (C) Mass chromatograms of SRM for base-related product ions of 2′-O-Me C (m/z 112; black line), 2′-O-Me U (m/z 113; green line), 2′-O-Me G (m/z 152; red line), and 2′-O-Me A (m/z 136; blue line). (D) piRNAs digested by RNase T2 were coinjected with a series of synthetic 3′-O-methyl nucleosides. Mass chromatograms for proton adducts of piRNA 3′-terminal nucleosides and a series of synthetic 3′-O-methyl nucleosides, 2′ or 3′-O-Me C (m/z 258; black line), 2′ or 3′-O-Me U (m/z 259; green line), 2′ or 3′-O-Me G (m/z 298; red line), and 2′ or 3′-O-Me A (m/z 282; blue line). Peaks for 3′-O-methyl nucleosides are indicated by arrowheads.
Figure 3.
Figure 3.
Involvement of CG12367 in piRNA methylation in fly ovary. (A) RT–PCR shows that the homozygous piggyBacf00810 mutant does not express CG12367 mRNAs, but does express transcripts of CG8878. A schematic drawing of CG12367 (blue) and CG8878 (red) genes in Drosophila is shown in Supplementary Figure S3A. RT–PCR reaction was performed using primers that are indicated in Supplementary Figure S3A. (−/+) PiggyBacf00810/CyO; (−/−) PiggyBacf00810/PiggyBacf00810; (wild type) yellow-white. (B) piRNAs associated with Aub and Piwi in the CG12367 mutant ovary (shown in A) were subjected to oxidation/β-elimination chemical reactions. In both cases, piRNAs gain mobility, indicating that piRNAs associated with Aub and Piwi in the CG12367 mutant are not modified at their 3′ ends.
Figure 4.
Figure 4.
GST-Pimet methylates single-stranded small RNAs in vitro. (A) miR-1/miR-1* duplexes (0.04 nmol/50 μL) were incubated with GST, GST-Pimet, and GST-HEN1 in the presence of 14C-labeled SAM. Resultant small RNAs were run on a denaturing acrylamide gel. The top panel (14C; autoradiograph of 14C-labeled RNAs) shows that only GST-HEN1 is able to transfer methyl groups from SAM to miRNA/miRNA* duplexes. The bottom panel shows a staining image (TBO, Toluidine Blue O) of the top gel, which indicates that the same amounts of miRNA/miRNA* duplexes were used in all the lanes. (B, top panel) When single-stranded small RNAs (26 nt) are the substrate for the methylation assay (0.5 nmol/50 μL), GST-Pimet shows stronger activity compared with that of GST-HEN1. The bottom panel shows a staining image of the top gel. (C) ssRNAs (22 nt, 26 nt, and 38 nt) were methylated by GST-Pimet in vitro. GST-Pimet seems to be indifferent to the length of substrates, unlike Arabidopsis HEN1 that hardly recognize 25-nt or longer RNAs as substrates in an in vitro methylation assay (Yang et al. 2006). (D) 2′-O-methyl modification of 26-nt ssRNAs prevents methylation, suggesting that only the 2′-hydroxyl group on the ribose of the far 3′ nucleotide is methylated by Pimet. (E) ssRNAs (26 nt) were methylated by GST-Pimet as in B and subjected to oxidation and β-elimination treatment. Only methylated ssRNAs (not visible in the bottom “TBO” panel) showed resistance (14C) to the chemical treatment.
Figure 5.
Figure 5.
(A) Pimet methylates piRNAs associated with Aub obtained from Pimet mutant ovary, but does not methylate miRNAs associated with AGO1. Aub–piRNA complexes were immunopurified from Pimet mutant ovary lysate with anti-Aub antibody (Gunawardane et al. 2007) and subjected to methylation by GST-Pimet. miRNAs associated with AGO1 immunopurified from Pimet mutant ovary lysate were used as a control. Nonimmune IgG (n.i.) was also used as a negative control. (Top panel) piRNAs were even methylated in a complex with PIWI protein (14C). By contrast, miRNAs associated with AGO1 were not methylated, although these miRNAs are single-stranded in a complex form with AGO1 (Miyoshi et al. 2005). (Bottom panel) miRNA levels appearing in A were several-fold higher than those of piRNAs (32P; 5′ end phosphorylation after CIP). (B) Aub–piRNA complexes immunopurified from Pimet ovary lysate were subjected to methylation by GST-Pimet as in A. piRNAs were then isolated and subjected to oxidation and β-elimination treatment. piRNAs methylated in vitro showed resistance to the chemical treatment. (C) Association of Pimet with PIWI proteins. GST-Pimet (Supplementary Fig. S4C) was incubated with the Pimet mutant ovary lysate, and after extensive washing the eluates were probed with antibodies against Aub, Piwi, AGO3, and AGO1. Aub, Piwi, and AGO3 are clearly detected in the bound fraction with GST-Pimet but not with GST. AGO1 was not observed in either lane.

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