Looking for a needle in a haystack: de novo phenotypic target identification reveals Hippo pathway-mediated miR-202 regulation of egg production

Abstract

AbstractUnderstanding microRNA (miRNA) functions has been hampered by major difficulties in identifying their biological target(s). Currently, the main limitation is the lack of a suitable strategy to identify biologically relevant targets among a high number of putative targets. Here we provide a proof of concept of successfulde novo(i.e., without prior knowledge of its identity) miRNA phenotypic target (i.e., target whose de-repression contributes to the phenotypic outcomes) identification from RNA-seq data. Using the medakamir-202knock-out (KO) model in which inactivation leads to a major organism-level reproductive phenotype, including reduced egg production, we introduced novel criteria including limited fold-change in KO and low interindividual variability in gene expression to reduce the list of 2,853 putative targets to a short list of 5. We selectedtead3b,a member of the evolutionarily-conserved Hippo pathway, known to regulate ovarian functions, due to its remarkably strong and evolutionarily conserved binding affinity for miR-202-5p. Deleting the miR-202-5p binding site in the 3’ UTR oftead3b, but not of other Hippo pathway memberssav1andvgll4b, triggered a reduced egg production phenotype. This is one of the few successful examples ofde novofunctional assignment of a miRNA phenotypic targetin vivoin vertebrates.