Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis

Abstract

Small RNAs of 21-25 nucleotides (nt), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), act as guide RNAs to silence target-gene expression in a sequence-specific manner. In addition to a Dicer homolog, DCL1, the biogenesis of miRNAs in Arabidopsis requires another protein, HEN1. miRNAs are reduced in abundance and increased in size in hen1 mutants. We found that HEN1 is a miRNA methyltransferase that adds a methyl group to the 3'-most nucleotide of miRNAs, but the role of miRNA methylation was unknown. Here, we show that siRNAs from sense transgenes, hairpin transgenes, and transposons or repeat sequences, as well as a new class of siRNAs known as trans-acting siRNAs, are also methylated in vivo by HEN1. In addition, we show that the size increase of small RNAs in the hen1-1 mutant is due to the addition of one to five U residues to the 3' ends of the small RNAs. Therefore, a novel uridylation activity targets the 3' ends of unmethylated miRNAs and siRNAs in hen1 mutants. We conclude that 3'-end methylation is a common step in miRNA and siRNA metabolism and likely protects the 3' ends of the small RNAs from the uridylation activity.

Figure 1. miRNAs in hen1 Have 3′-End Defects

(A) RNA filter hybridization of miR167 (21 nt), miR172 (21 nt), miR173 (22 nt), miR163 (24 nt), and miR171 (21 nt) in various genetic backgrounds. Ler is the wild-type control for hen1-1, hen1-2, hen1-3, and dcl1-9. Col is the wild-type control for hen1-4, hen1-6 (SALK_090960), and dcl3-1. (B) 5′ ends of miRNAs in hen1-1 are the same as those in Ler as detected by primer extension. Primer extension was performed as described [24], with 17–18 nt DNA primers spanning the 3′ portions of mature miRNAs. The extension products are indicated by the arrowheads. (C) Accumulation of miR172 in various genotypes as detected by RNA filter hybridization. (D) Detection of 3′ methylation of miR171. Total RNAs were treated (+β) or not (−β) with the oxidation and β elimination reagents, and miR171 was detected by RNA filter hybridization. miR171 from the wild-type is resistant, whereas the heterogeneous miR171 species in hen1-1 are sensitive to the chemical reactions. In (A), (C), and (D), images of ethidium-bromide-stained gels in the region of tRNAs are shown at the bottom to indicate near-equal loading.

Figure 2. miRNAs in hen1-1 Are Uridylated at Their 3′ Ends

(A) A schematic diagram of an α-³²P-dATP incorporation assay. The first three residues in the 3′ adaptor are T (red). T residues between the mature miRNAs (black line) and the 3′ adaptor (red line and letters) in the RT-PCR products are in green. The primer complementary to the 3′ adaptor (excluding the three T residues) is in blue. (B) α-³²P-dATP incorporation assay performed on miR173, miR389a1, miR399f, and miR173* from Ler and hen1-1. The bottom three bands marked by the bars represent the extension products corresponding to the Ts in the 3′ adaptor. Extension products marked by the brackets indicate the T residues between the mature miRNAs and the 3′ adaptor.

Figure 3. A 24 nt Endogenous siRNA in hen1 Is Unmethylated and 3′ Uridylated

(A) Accumulation of siRNA02 in various genotypes as detected by RNA filter hybridization. (B) Detection of 3′ methylation of siRNA02. Total RNAs were treated (+β) or not (−β) with the oxidation and β elimination reagents, and siRNA02 was detected by filter hybridization. siRNA02 is methylated in Ler and unmethylated in hen1-1. Images of ethidium-bromide-stained gels in the region of tRNAs in (A) and (B) are shown at the bottom to indicate near-equal loading. (C) The 5′ ends of siRNA02 in hen1-4 and hen1-6 are the same as those in the wild-type as detected by primer extension. A 15 nt DNA primer complementary to the 3′ portion of siRNA02 was used. (D) U tails at the 3′ ends of siRNA02 in hen1-1 were detected by an α-³²P-dATP incorporation assay (diagrammed in Figure 2A). The bottom three bands marked by the bars represent the extension products corresponding to the three Ts in the 3′ adaptor. Extension products marked by the brackets indicate the T residues between the mature siRNA and the 3′ adaptor. The sequence of siRNA02 is shown. The presence of an extension product 1 nt beyond the 3′ adaptor in the wild-type is probably due to the presence of some siRNA02 species ending with the penultimate T nucleotide.

Figure 4. siRNAs Produced from Transgenes and a ta-siRNA Are Methylated in the Wild-Type but Not in hen1

(A) Accumulation of S-PTGS siRNAs from GUS transgene (L1 locus) in the wild-type (Col) before and after the oxidation and β elimination reactions (+β) and in hen1-4 and hen1-6 (SALK_090960). (B) Accumulation of IR-PTGS siRNAs from an AP1 hairpin transgene in the wild-type (Col) and hen1-4 before and after (+β) the oxidation and β elimination reactions. (C) Accumulation of a ta-siRNA in the wild-type (Ler) and hen1-1 before and after (+β) the oxidation and β elimination reactions. Images of ethidium-bromide-stained gels in the region of tRNAs are shown at the bottom.