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Abstract

MicroRNAs (miRNAs) are small regulatory RNAs of 19 to 22 nucleotides in length, which regulate gene expression at the posttranscriptional level. In contrast to the mechanism of translational inhibition proposed for animal miRNAs, plant miRNAs mostly target transcripts for cleavage, requiring extensive base paring between the miRNA and target transcript. Previous studies have revealed the importance of miRNA-Jaw (miR319a) in plant development. Ectopic miR319a expression in jaw-D plants causes pleiotropic developmental defects, including epinastic cotyledons, crinkly leaves, and a modest delay in flowering time. MiRNA-Jaw negatively regulates transcript levels of five TCP transcription factors that are thought to be involved in the regulation of cell proliferation. We carried out a suppressor screen to analyze the miR319a/TCP interaction in vivo, and to identify other factors necessary for the phenotypic effects of ectopic miR319a expression. 16 potential suppressor mutants were identified, called suppressor of jaw-D, (soj). These affect the miR319a pathway at different levels. Here we focus on four point mutations that impair the miR319a guided cleavage of the TCP4 transcript. Three of these mutations are located within the miR319a target site of TCP4, whereas one was found in the mature miR319a. We show that all mutations partially impair the efficiency of miR319a guided cleavage of TCP4. Although the mutant TCP4 alleles have no dramatic effects in the endogenous genomic context, their overexpression in transgenic plants leads to severe defects in early plant development. In summary, suppressor mutagenesis of miRNA overexpressers is an efficient tool for studying miRNA-target interaction in vivo.