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.

Asymmetric bulges and mismatches determine 20-nt microRNA formation in plants

“…To test this possibility, we carried out b-elimination treatment after which unmethylated miRNAs run faster than methylated miRNAs due to the loss of the terminal nucleotide. 46 As expected, athmiR156c migrated faster in the hen1-1 mutant (Fig. S5).…”

“…46 Low molecular weight RNA of P. patens grown on solid BCDAT medium at 25 C under continuous white light was isolated using mirVana miRNA Isolation Kit (Ambion). One to 8 micrograms of P. patens small RNA was dissolved in 70 ml borax/boric acid buffer, pH 8.6 (4.375 mM borax, 50 mM boric acid) and 10 ml 0.2 M sodium periodate (Sigma) was added.…”

HUA ENHANCER1 Is Involved in Posttranscriptional Regulation of Positive and Negative Regulators in Arabidopsis Photomorphogenesis

“…We generated transgenic Arabidopsis carrying a HA-tagged HEN1 (HA-HEN1) under the control of a bestradiol inducible promoter (XVE:HA-HEN1) (Zuo et al, 2006). HA-HEN1 was previously shown to be a biologically active protein (Yang et al, 2007). We first induced HA-HEN1 expression in etiolated seedlings of transgenic plants under dark conditions to eliminate the effect of light-upregulated HEN1.…”

“…HY5 protein was detected by polyclonal anti-HY5 antibody (Liu et al, 2012). HAtagged HEN1 protein was detected by incubation with the antibodies mouse monoclonal anti-HA (H3663; Sigma-Aldrich) or pig polyclonal anti-HEN1 (Yang et al, 2007). Endogenous a-tubulin was detected by incubation with a mouse monoclonal anti-a-tubulin antibody (T5168; Sigma-Aldrich).…”

miR-ID: A novel, circularization-based platform for detection of microRNAs

“…miR-ID is an attractive alternative to the commonly used periodate-oxidation method for identifying 29-O-methylation at the 39-end of small RNAs (Yang et al 2007). That method has several disadvantages: (1) It is laborious; (2) it requires gel-electrophoresis analysis, which offers comparatively low sensitivity and is semi-quantitative; and (3) it is not specific to individual miRNAs.…”