Suppression of microRNA accumulation via RNA interference in Arabidopsis thaliana

Abstract

MicroRNAs (miRNAs) are key regulatory molecules in plants. These small RNAs are processed in the nucleus from longer precursor transcripts that form distinct secondary structures. The miRNAs target specific messenger RNAs (mRNAs) and consequently down-regulate gene expression. The importance of these regulatory molecules is wide-ranging, however, few loss-of-function mutants have been identified in miRNA genes and understanding the biology of miRNA-target pairings has largely depended upon creating alterations in the sequences of the target genes. Here we demonstrate using Arabidopsis thaliana, that it is possible to use RNA interference (RNAi) to suppress accumulation of miRNAs. Significantly reduced accumulation of miR163 and miR171a was achieved using hairpin RNAi constructs that were designed to target both the primary miRNA transcripts and their promoters. The presence of DNA methylation in the targeted promoter regions suggests that inhibition of transcription of the miRNA precursors is responsible. Reduction of miRNA accumulation resulted in an increase in accumulation of the mRNA targets of these miRNAs. This demonstrates that knock-down of miRNA expression can be achieved, thereby providing a straightforward approach for disrupting miRNA-target pairings and studying miRNA functions.