Global identification of microRNA–target RNA pairs by parallel analysis of RNA ends

Abstract

The targets of a microRNA (miRNA) are usually identified by computational analysis of sequences complementary to the miRNA. Working with inflorescence tissue of Arabidopsis, German et al. devise an experimental approach in which the products of miRNA-mediated cleavage are sequenced and used to identify miRNA–target RNA pairs. MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5′ ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA–target RNA pairs. Within the set of ∼27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5′-to-3′ exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA–target RNA pairs.