Approaches for studying microRNA and small interfering RNA methylation in vitro and in vivo

Abstract

The biogenesis of microRNAs (miRNAs) in plants is similar to that in animals, however, the processing of plant miRNAs consists of an additional step, the methylation of the miRNAs on the 3' terminal nucleotides. The enzyme that methylates Arabidopsis miRNAs is encoded by a gene named HEN1, which has been shown genetically to be required for miRNA biogenesis in vivo. Small interfering RNAs (siRNAs) are also methylated in vivo in a HEN1-dependent manner. Our biochemical studies demonstrated that HEN1 is a methyltransferase acting on both miRNAs and siRNAs in vitro. HEN1 recognizes 21 to 24 nt small RNA duplexes, which are the products of Dicer-like enzymes, and transfers a methyl group from S-adenosylmethionine (SAM) to the 2' OH of the last nucleotides of the small RNA duplexes. Here we describe methods to characterize the biochemical activities of the HEN1 protein both in vitro and in vivo, and methods to analyze the methylation status of small RNAs in vivo.

Figure 8.1

SDS-polyacrylamide gel electrophoresis of affinity-purified recombinant GST-HEN1 (lane1) and His₆-HEN1 (lane 2). Protein staining with Coomassie Brilliant Blue R-250 indicates a greater than 90% purityof both recombinant methyltransferases. The sizes of molecular mass markers are indicated on the left.

Figure 8.2

A time course of dsRNA methylation catalyzed by the His₆-HEN1 methyltransferase. Experiment was performed at 37° in Reaction Buffer containing 2-μM miR173/miR173★ RNA, 20-μM [methyl-³H]-SAM (4.7 Ci/mmol), and 0.1-μM methyltransferase. Duplicate aliquots were removed for analysis at specified time points and processed as described.

Figure 8.3

β-elimination of an RNA oligonucleotide with two free OH groups on the 3′ terminal ribose. The RNA was treated (+) or untreated (−) with the chemicals for β-elimination, resolved on a 15% polyacrylamide gel with urea, blotted to a membrane, and hybridized with an antisense probe.

Figure 8.4

Immunoprecipitation (IP) of HEN1 followed by a methyltransferase assay. IP was performed from inflorescence tissues of 35S∷HA-HEN1 hen1–1 (lane1) or hen1–1 (lane 2). A, Western blotting with anti-HEN1 polyclonal antibodies. Ten-μl beads from the IP were mixed with 10-μl 2× SDS sample buffer, boiled, and loaded on a 10% SDS-polyacrylamide gel for Western blotting with anti-HEN1 polyclonal antibodies. Sizes of molecular mass markers are indicated on the left. B, A HEN1 methyltransferase assay with the immunoprecipitates from 35S∷HA-HEN1 hen1–1 (lane 1) and hen1–1 (lane 2). The miR173/miR173★ duplex was used as the substrate and [methyl-¹⁴C] SAM was used as the cofactor.