RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse

Abstract

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on <i>Rnf212</i> but not on <i>Msh4</i>, <i>Hei10,</i> and <i>Cntd1</i>, while the unloading of RNF212B at the end of pachynema is dependent on <i>Hei10</i> and <i>Cntd1</i>. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for <i>Rnf212b</i> and <i>Rnf212</i> exhibit an identical phenotype to that of <i>Rnf212b</i> single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from <i>Rnf212b</i> mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.