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. 2005 Feb 11;307(5711):932-5.
doi: 10.1126/science.1107130.

Methylation as a crucial step in plant microRNA biogenesis

Bin Yu  1 , Zhiyong Yang, Junjie Li, Svetlana Minakhina, Maocheng Yang, Richard W Padgett, Ruth Steward, Xuemei Chen
Affiliations

Methylation as a crucial step in plant microRNA biogenesis

Bin Yu et al. Science. .

Abstract

Methylation on the base or the ribose is prevalent in eukaryotic ribosomal RNAs (rRNAs) and is thought to be crucial for ribosome biogenesis and function. Artificially introduced 2'-O-methyl groups in small interfering RNAs (siRNAs) can stabilize siRNAs in serum without affecting their activities in RNA interference in mammalian cells. Here, we show that plant microRNAs (miRNAs) have a naturally occurring methyl group on the ribose of the last nucleotide. Whereas methylation of rRNAs depends on guide RNAs, the methyltransferase protein HEN1 is sufficient to methylate miRNA/miRNA* duplexes. Our studies uncover a new and crucial step in plant miRNA biogenesis and have profound implications in the function of miRNAs.

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Figures

Fig. 1
Fig. 1
HEN1 has miRNA methyltransferase activity in vitro. The RNA substrates were incubated with the proteins and resolved by electrophoresis, and the presence of 14C-labeled RNA was detected by autoradiography. (A) miRNA methyltransferase assay using the miR173/miR173* duplex as the substrate. (B) Various RNA substrates were tested for methylation by GST-HEN1. The substrates used are the putative pre-miR173 (lane 1), miR173/miR173* duplex (lane 2), miR173 (lane 3), miR173* (lane 4), a DNA duplex identical in sequence to miR173/miR173* (lane 5), E. coli tRNA (lane 6), and miR167a/miR167a* duplex (lane 7). Lane 8, miR173/miR173* was incubated with GST.
Fig. 2
Fig. 2
Methylation of miRNA/miRNA* in vitro requires the two free OH groups on the 3′-most nucleotides. The substrates diagrammed in (A) were assayed for methylation by GST-HEN1 in (B). The numbers of the molecules in (A) correspond to the lane numbers in (B). In the diagrams, the top strand is miR173 (22 nt), which is 1 nt longer than miR173*, the bottom strand. The two mismatches in the duplex are represented by the bulges. Species 2 in (A) differs from species 1, in that the miR173m1* strand is longer by 2 nt at its 5′ end but shorter by 2 nt at its 3′ end than miR173*. In species 3 to 8, d1 and d2 represent a deoxyribose at the 2′ carbon of the last and penultimate nucleotide, respectively. Species 8 has a deoxyribose at the 3′ carbon of the last nucleotide.
Fig. 3
Fig. 3
One or both OH groups on the ribose of the last nucleotide of Arabidopsis miRNAs are blocked. Total RNAs were treated with periodate followed by β elimination and then subjected to RNA filter hybridization with probes against various miRNAs (as indicated next to the blots). The region of the stained gel corresponding to where tRNAs migrate is shown below the hybridization images to indicate the amount of total RNAs used. (A) In vitro synthesized miR173-OH or miR173-2′me was added to total RNA from dcl1-9 (d) and treated with the chemicals. miR173-OH showed the expected mobility shift but miR173-2′me was resistant to the chemical modification reactions. (B) miR173 from wild-type total RNA (w) is resistant to the chemical modification reactions. (C) Total RNA from wild type (wt) or hen1-1 was either subjected (β) or not (−) to the chemical modification reactions and probed for various miRNAs.
Fig. 4
Fig. 4
Mass spectrometry analysis of miR173 isolated from Arabidopsis (A) and a miR173 standard with one methyl group (B). The position of a peak along the x axis represents the molecular mass of the species. Signal intensity approximately reflects the amount of the RNA in each peak. The expected mass of the standard is indicated by the number in parentheses. The measured mass is indicated by the numbers directly above the peaks.
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