Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase

Abstract

Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3′UTR with two regions of AID-dependent Ago2-binding sites (5′- and 3′dABs). Top1 3′UTR knockout (3′UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3′UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3′UTR–mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5′- and 3′dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3′UTRKO cells to wild-type cells whereas it does not impact on 3′UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3′UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.