Identification of new RAD51D-regulating microRNAs that also emerge as potent inhibitors of the Fanconi anemia/homologous recombination pathways

The Fanconi anemia (FA) and homologous recombination (HR) pathways, which partially overlap and include RAD51 and its paralogs, are key for the repair of different types of DNA damage, such as DNA interstrand crosslinks. First, to broadly assess the impact of microRNA-mediated regulation, we examined microRNA expression profiles in five isogenic fibroblast cell pairs, either deficient in DNA repair due to germline mutations in FANCA, FANCB, FANCC, FANCI or BRIP1/FANCJ or proficient due to correction with retroviral vectors. In each pair, we observed lower abundance of specific microRNAs in the FA-deficient cells. From the list of microRNAs, we experimentally confirmed the effects of miR-141-3p and miR-369-3p targeting RAD51B and miR-15a-5p, miR-494-3p as well as miR-544a targeting RAD51D. However, by western blotting, only RAD51D protein was reduced by a mixture of its regulating microRNAs. Gene ontology analyses and identification of additional FA/HR factors as targets of miR-15a-5p, miR-494-3p and miR-544a strongly suggested the widespread influence of these microRNAs on HR. Interestingly, only miR-494-3p directly reduced RAD51 foci formation, while a mixture of miR-15a-5p, miR-494-3p and miR-544a strongly reduced HR activity in green fluorescent protein (GFP) repair assays. In summary, by successfully employing this novel loss- and gain-of-function strategy, we have identified new microRNAs strongly inhibiting HR in mammalian cells. Understanding and modulating such miRNA regulation of DNA repair genes/pathways might help to overcome the reduced repair capacity of FA patients with biallelic hypomorphic mutations or help to engineer synthetic lethality strategies for patients with mutations in cancer-associated FA/HR genes.