Mammalian Oocytes Store mRNAs in a Mitochondria-Associated Membraneless Compartment

Abstract

ABSTRACT During the final stages of oocyte growth, transcription ceases only to resume following fertilization. During this cessation, the oocyte and the embryo can only use stores mRNAs to synthesize new products, meaning the storage of maternal mRNAs is critical for generating haploid eggs through meiosis and for early embryonic development. Membraneless storage compartments are well characterized in nonmammalian oocytes but are less understood in more relevant models. Studies have established that RNA-binding proteins YBX2 and ZAR1 are required for maternal mRNA storage in full-grown mouse oocytes, and this study aimed to further characterize the localization of maternal mRNAs and the mechanism that stores them. The potential colocalization between highly expressed RNA-binding proteins and different types of membrane-bound organelles in fully grown mouse oocytes was analyzed, finding the proteins ZAR1, YBX2, DDX6, LSM14B, and 4E-T colocalized with mitochondria. Similar results were found in human, porcine, and bovine oocytes. The localization domain of RNA-binding proteins around mitochondria throughout the cytoplasm was named the mitochondria-associated ribonucleoprotein domain (MARDO). Immunostaining for ZAR1 at various growth stages was performed to determine when the MARDO forms, revealing that formation is directed by an increase in mitochondrial membrane potential during oocyte growth. ZAR1 was observed to be the most highly enriched of these proteins on mitochondria and to be the promoter of MARDO coalescence and mitochondrial clustering. Injections of RNase into mouse oocytes revealed MARDO is a phase-separated compartment regulated by RNA levels, and further analysis identified the disordered N-terminal region of ZAR1 drives MARDO coalescence into a hydrogel-like matrix that sequesters mitochondria. Next, the degradation of MARDO at the meiosis stage when oocytes resume maturation was analyzed. ZAR1 was found to be progressively depleted from the mitochondria during oocyte meiotic maturation. Mass spectrometry revealed phosphorylation as a driving mechanism for this disassembly during maturation. MARDO dissolution accompanied a decrease in ZAR1 levels and occurred during the transition from MI to MII. Treating oocytes with a proteasome inhibitor blocked dissolution of MARDO, whereas impairing spindle assembly causing cell-cycle arrest before MI had no effect. This suggested that proteasome-mediated degradation of ZAR1 underlies MARDO dissolution during oocyte meiotic maturation. Knockout studies in mice confirmed that ZAR1 is essential for MARDO assembly, and in these knockouts more than 1000 mRNAs, many of which were enriched in the MARDO, were reduced by at least 50%, suggesting MARDO disruption also causes mRNA reduction. Many RNA-binding proteins in the MARDO are implicated in translational repression, and by labeling translating ribosomes assembled on mRNAs, it was seen that these active ribosomes were mostly excluded from the MARDO. In addition, ZAR1 was seen to substantially reduce the translation of bound mRNA, suggesting that the maternal mRNAs stored in the MARDO are translationally repressed. This study identified and characterized the MARDO, a mitochondria-associated membraneless compartment that stores maternal mRNAs in mouse, bovine, porcine, and human oocytes.