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Transgenically Expressed Viral RNA Silencing Suppressors Interfere with Microrna Methylation in Arabidopsis

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Transgenically Expressed Viral RNA Silencing Suppressors Interfere with Microrna Methylation in Arabidopsis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 580 (2006) 3117–3120 Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis Bin Yua, Elisabeth J. Chapmanb, Zhiyong Yanga, James C. Carringtonb, Xuemei Chena,* a Department of Botany and Plant Sciences and Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, USA b Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA Received 12 March 2006; revised 19 April 2006; accepted 23 April 2006 Available online 2 May 2006 Edited by Shou-Wei Ding on miRNA/miRNA* duplexes in vivo. It is also unknown Abstract HEN1-dependent methylation of the 30-terminal nucleotide is a crucial step in plant microRNA (miRNA) biogen- where in the cell miRNA methylation occurs. esis. Here we report that several viral RNA silencing suppressors Most plant viruses encode RNA silencing suppressors, which (P1/HC-Pro, p21 and p19) inhibit miRNA methylation. These condition susceptibility by interfering with the host antiviral suppressors have distinct effects on different miRNAs. We also silencing response. Many viral RNA silencing suppressors are show that miRNA* is methylated in vivo in a suppressor-sensitive also pathogenicity factors that cause developmental defects in manner, suggesting that the viral proteins interfere with miRNA/ host plants [10]. The miRNA pathway is inhibited by several miRNA* duplexes. p19 and p21 bind both methylated and unme- viral silencing suppressors [11–13], such as the Beet yellows * thylated miRNA/miRNA duplexes in vivo. These findings sug- virus 21 kDa protein (p21) [14], the Tomato bushy stunt virus * gest miRNA/miRNA as the in vivo substrates for the HEN1 19 kDa protein (p19) [15,16] and the Turnip mosaic virus silenc- miRNA methyltransferase and raise intriguing possibilities ing suppressor, P1/HC-Pro [17–19]. Each of these suppressors regarding the cellular location of miRNA methylation. * Ó 2006 Federation of European Biochemical Societies. Published stabilizes miRNA/miRNA duplexes [12,13]. Although P1/ by Elsevier B.V. All rights reserved. HC-Pro was initially thought to stabilize duplexes by an indi- rect mechanism [12], recent studies using in vitro and in vivo as- Keywords: Viral RNA silencing suppressor; Methylation; says revealed that all three suppressors interact directly with microRNA/microRNA*; HEN1 small RNA duplexes [20]. Since P1/HC-Pro, p21 and p19 act on small RNA duplexes, they may affect miRNA methylation if miRNA/miRNA* duplexes are the in vivo substrates of HEN1. Recently it was shown that the expression of P1/HC- Pro weakly affects the modification of miRNAs in tobacco [5]. 1. Introduction Here we report that viral RNA silencing suppressors p21, p19 and P1/HC-Pro interfere with the methylation of MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) RNAs miRNAs. that function as sequence-specific regulators of gene expression through translational repression and/or transcript cleavage [1]. 2. Materials and methods miRNA/miRNA* duplex intermediates are formed by DICER 0 or DICER-like enzymes, which generate products with 2-nt 3 2.1. Transgenic plants overhangs containing 50 monophosphate and 30-OH groups. Arabidopsis thaliana Col-0 transgenic plants expressing influenza The miRNA strand is incorporated into the RNA-induced HA epitope-tagged P1/HC-Pro,p21, p19 or the empty expression vec- silencing complex, while the passenger miRNA* strand is tor were constructed as described [12]. Seeds were grown under selec- tion for phosphoinothricin resistance and pools of primary ejected and degraded [1]. We and others recently demonstrated transformants were analyzed. that the 20-OH of the 30-terminal nucleotide of miRNAs and siRNAs is methylated in Arabidopsis by a methyltransferase, 2.2. Detection of small RNAs by RNA filter hybridization HUA ENHANCER1 (HEN1) [2–5]. The reduced accumula- RNA isolation, gel electrophoresis, blotting, hybridization and tion and heterogeneity in size of miRNAs, as well as loss of washes for miRNAs and 5S RNAs were performed as described [6]. 32 miRNA function, in hen1 mutants [6–9] reflect the importance 50 end-labeled ( P) antisense DNA or LNA oligonucleotides were of miRNA methylation in plants. The size increase of small used as probes. Radioactive signals were detected and quantified with a phosphorimager. RNAs in the hen1-1 mutant is due to the addition of one to five 0 U residues to the 3 ends of the small RNAs by a novel uridy- 2.3. Analysis of the methylation status of small RNAs 0 lation activity targeting the 3 ends of unmethylated miRNAs Sodium periodate treatments were done as described [2,21,22]. RNA and siRNAs [3]. Therefore, one role of small RNA methyla- was dissolved in borax/boric acid buffer (0.06 M, pH 8.6) and sodium tion is likely protecting 30 ends of the small RNAs from uridy- periodate (200 mM in water) was added to a final concentration of lation activity. HEN1 uses miRNA/miRNA* duplexes as 25 mM. The RNA was then incubated in darkness at room tempera- ture. After 1 h of incubation, 1/10 volume of glycerol was added to substrates in vitro [2,4], but it is not known whether it also acts the RNA and the incubation was continued for an additional 30 min. The RNA was then precipitated in the presence of ethanol. For b elimination, the sodium periodate-treated RNA was dissolved *Corresponding author. Fax: +1 951 827 4437. in NaOH/borax/boric acid buffer (0.055 M, pH 9.5), incubated at E-mail address: [email protected] (X. Chen). 45 °C for 90 min, and precipitated with ethanol. 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.04.063 3118 B. Yu et al. / FEBS Letters 580 (2006) 3117–3120 2.4. Co-immunoprecipitation during electrophoresis by between one and two nucleotides The immunoprecipitation of HA-tagged viral RNA silencing sup- after the treatment. Methylated miRNAs whose 30 terminal ri- pressors and RNA isolation from precipitates were performed as de- bose contains a 20-O-methyl group do not participate in the scribed [12]. Briefly, tissues from transgenic Arabidopsis were ground in liquid nitrogen and homogenized in 5 ml/g lysis buffer (50 mM reactions and remain unchanged in mobility. This method 0 Tris–HCl at pH 7.4, 100 mM KCl, 2.5 mM MgCl2, 0.1% NP-40, and was previously used to study miRNA 3 end ribose methyla- 2· complete protease inhibitor cocktail; Roche), and centrifuged for tion [2,22]. Although this method does not distinguish a 20- 15 min at 9500 · g. Clarified lysates were pre-cleared with protein A- O-methyl modification from other potential modifications on agarose, reacted with anti-HA (Roche) for 1 h at 4 °C, and then incu- 0 bated with protein A-agarose beads for 3 h at 4 °C. Precipitates were the 3 terminal ribose, previous mass spectrometry studies washed three times in lysis buffer and divided for protein and RNA demonstrated that methylation is the only modification of an analysis. Nucleic acid was recovered by treatment with 3 volumes of Arabidopsis miRNA [2]. We used this method to evaluate 30 proteinase K solution (100 mM Tris–HCl, pH 7.4, 10 mM EDTA, terminal methylation of miRNAs in the RNA silencing sup- 150 mM NaCl, 2% SDS, and 0.2 lg/ll proteinase K) for 15 min at pressor-expressing plants. The methylation status of miR159, 65 °C followed by extraction with saturated phenol and phenol:chloro- form and ethanol precipitation. miR167, miR171 and miR172 was analyzed in pools of pri- mary transformants containing each of the three viral silencing suppressors or the vector DNA alone [12]. As shown in 3. Results Fig. 1A, a portion of the miRNAs from the suppressor- expressing plants migrated more quickly after b elimination 3.1. Viral silencing suppressors inhibit miRNA methylation reactions, except for miR171 from p21 transgenic lines. No To determine if p19, p21 and P1/HCPro affect the methyla- mobility change was detected in the vector-transformed plants, tion of the 30-terminal nucleotide of miRNAs, total RNA from indicating that the miRNA pools in control plants were fully Arabidopsis transgenic lines that constitutively expressed these or near fully methylated. The proportion of unmethylated viral silencing suppressors [12] was treated with sodium perio- miRNAs ranged from 17.5% (miR171) to 42.7% (miR159) in date followed by b elimination. Specific miRNAs were ana- p19-expressing plants, 19.5% (miR159) to 68% (miR172) in lyzed by RNA filter hybridization. The reactions eliminate a P1/HC-Pro-expressing plants and from undetectable 30 nucleotide containing both 20 and 30 OH groups on the ri- (miR171) to 88.5% (miR172) in p21-expressing plants bose, resulting in an RNA product that is one nucleotide (Fig. 1B). Therefore, the viral silencing suppressors partially shorter than the substrate RNA and that contains a 30 phos- affected miRNA methylation, but to different levels depending phate group [21]. Therefore, miRNAs that contain a 30 termi- on the miRNA. The previously observed truncated forms of nal ribose with both 20 and 30 OH groups will migrate faster miRNAs in p19 transgenic plants [12,13,23] were detected Fig. 1. The viral silencing suppressors inhibit miRNA methylation. (A) The methylation status of miRNAs in various transgenic lines. Total RNAs from vector-transformed or viral silencing suppressor-transformed plants were either subjected (+) or not (À) to the chemical modification reactions and probed for various miRNAs by filter hybridization. The region of the stained gels corresponding to where tRNAs migrate is shown below the hybridization images to indicate the amount of total RNAs used.
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