Computational and experimental analysis of plant microRNAs

Abstract

MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that mediate gene regulation in plants and animals. We demonstrated that Arabidopsis thaliana miRNAs are highly complementary (0-3 mispairs in an ungapped alignment) to more mRNAs than would be expected by chance. These mRNAs are therefore putative regulatory targets of their complementary miRNAs. Many miRNA complementary sites are conserved to the monocot Oryza sativa (rice), implying evolutionary conservation based on function at the nucleotide level. The majority of predicted miRNA targets encode for transcription factors and other proteins with known or inferred roles in developmental patterning, implying that the miRNAs themselves are high-level regulators of development. Our findings indicated that miRNAs are key components of numerous regulatory circuits in plants and set the stage for numerous additional experiments to investigate in depth the significance of miRNA-mediated regulation for particular target families and genes. We developed a comparative genomics approach to identify miRNAs and miRNA targets conserved between Arabidopsis and Oryza. Seven previously unknown miRNAs families were experimentally verified, bringing the total number of known miRNA genes in Arabidopsis to 92, representing 22 families. We expanded the range of functionalities known to be regulated by miRNAs to include F-box proteins, laccases, superoxide dismutases, and ATP-sulfurylases. The expression of miR395, which targets sulfate metabolizing enzymes, is induced by sulfate- starvation, demonstrating that miRNA expression can be responsive to growth conditions.

(cont.) We investigated the biological role of miR394-mediated regulation of Atlg27340, an F-box gene of previously unknown function. Transgenic plants expressing a miR394-resistant version of Atlg27340 displayed a range of developmental abnormalities, including radialized and fused cotyledons, absent shoot apical meristems, curled and radialized leaves, and abortive flowers. The severity of these abnormalities correlated with the overaccumulation of Atlg27340 mRNA. These findings confirm the biological relevance of the interaction between miR394 and Atlg27340, and represent the first insights into the roles of miRNA-mediated regulation of F-box genes. Our results establish that both MIR394 and Atlg27340 are important regulators of meristem identity, and suggest that Atlg27340 targets an activator of class III HD-ZIP function for ubiquitination and proteolysis.