Target Validation of Plant microRNAs

Abstract

microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate mRNAs that result in translational inhibition, mRNA decay, or slicing within the region of complementarity. miRNA processing activity on complementary target mRNAs generates 3′ end cleavage products that contain ­ligation-competent, 5′-monophosphates. Precise mapping of miRNA-directed cleavage sites within target transcripts is, therefore, possible using RNA ligase-mediated 5′ amplification of cDNA ends (RLM-RACE). Here, we provide a comprehensive RLM-RACE-based protocol for the amplification of 5′ ends derived from cleaved transcripts resulting from miRNA-guided cleavage events. Novel strategies for high-throughput analysis of miRNA cleavage products have emerged as powerful tools for the novo identification of miRNA targets in a genomic perspective. In this work, we also describe a novel methodology for genome-wide identification of miRNA targets that exploits RLM-RACE for non-sequence-specific enrichment of cleaved transcripts, T7 RNA polymerase-mediated amplification of target products, and microarray hybridization.