Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis

Abstract

HEN1-dependent methylation of the 3'-terminal nucleotide is a crucial step in plant microRNA (miRNA) biogenesis. Here we report that several viral RNA silencing suppressors (P1/HC-Pro, p21 and p19) inhibit miRNA methylation. These suppressors have distinct effects on different miRNAs. We also show that miRNA* is methylated in vivo in a suppressor-sensitive manner, suggesting that the viral proteins interfere with miRNA/miRNA* duplexes. p19 and p21 bind both methylated and unmethylated miRNA/miRNA* duplexes in vivo. These findings suggest miRNA/miRNA* as the in vivo substrates for the HEN1 miRNA methyltransferase and raise intriguing possibilities regarding the cellular location of miRNA methylation.

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 (−) 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. (B) Percentage of unmethylated miRNAs in various transgenic lines. Radioactive signals from (A) were quantified with a phosphorimager. The percentage was calculated as: {unmethylated miRNA/(unmethylated miRNA + methylated miRNA)} * 100%. The truncated miRNAs in p19 and HC-Pro lines were not included. (C) The methylation of miR172b* in various transgenic lines. Although the identities of the three species are unknown, they are all methylated in the vector-transformed plants.

Fig. 2

Co-immunoprecipitation (co-IP) of methylated and unmethylated miRNAs with the viral silencing suppressors. RNAs from the IP input (IN) sample or from the immunoprecipitates were either subject (+) or not (−) to the chemical modification reactions and probed for various miRNAs. The miR172 and miR171 panels were from the same blot and share the 5S rRNA control (bottom panel).