HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide

Abstract

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing.

Figure 1

In vitro methyltransferase reactions by GST-HEN1 on miRNA/miRNA* duplexes. Various RNA duplexes were methylated (see Materials and Methods) in the presence of [¹⁴C]-SAM. The RNAs were then resolved on polyacrylamide gels, which were exposed to X-ray films to obtain the autoradiograms. The numbers of the duplexes above the lanes correspond to the ones in Table 1. Since the activity of GST-HEN1 varies between protein preparations, assays performed with the same preparation of GST-HEN1 are on the same autoradiogram and are comparable to one another. Signals from (a) cannot be compared to those in (b).

Figure 2

In vitro methyltransferase reaction by GST-HEN1 on siRNA/siRNA* duplexes and miRNA/miRNA* duplexes with 1–5 3′ overhangs. All duplexes in this figure were methylated with the same GST-HEN1 preparation and are therefore comparable to one another. The numbers above the lanes correspond to those in Table 1.

Figure 3

Determination of the position of the methyl group by HPLC. (a) An elution profile of nucleoside standards. A, C, G, U, C-2′OMe and C-3′OMe were mixed and applied to the column. The retention times of C, G and U were determined by running each standard individually under identical conditions. The retention time of A was much longer than 20 min and is not shown. The retention times of C-3′OMe and C-2′OMe were determined by spiking the mix with more of either nucleoside in a separate run and noting which peak increased in amount. (b) An elution profile of the control methylation reaction with GST alone. (c) An elution profile of the methylation reaction with GST-HEN1. See ‘Materials and Methods’ for the treatment of the RNA duplexes before HPLC. The peak marked by an asterisk is the methyl-C generated by GST-HEN1. (d–f) Magnified versions of (a–c) in the region of 13–20 min.

Figure 4

Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. (a) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. (b) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α-³²P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. (c) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α-³²P]-ATP.