Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression. As miRNAs are involved in a wide range of biological processes and diseases, much effort has been invested in identifying their mRNA targets. Here we present a novel combinatorial approach, RIPchipSRM (RNA binding protein immunopurification + microarray + targeted protein quantification via selected reaction monitoring), to identify de novo highconfidence miRNA targets in the nematode Caenorhabditis elegans. We used differential RIP-chip analysis of miRNA induced silencing complexes from wildtype and miRNA mutant animals, followed by quantitative targeted proteomics via selected reaction monitoring to identify and validate mRNA targets of the C. elegans bantam homologue miR58. Comparison of total mRNA and protein abundance changes in mir58 mutant and wildtype animals indicated that the direct bantam/miR58 targets identified here are mainly regulated at the level of protein abundance, not mRNA stability.