Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment

Abstract

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

Oocytes store mRNAs around mitochondria

Mammalian oocytes stop transcribing DNA into messenger RNA (mRNA) during the final stages of their development. The oocyte’s meiotic divisions and early embryo development occur in the absence of transcription and rely instead on maternal mRNAs that are stored in the oocyte. However, where and how mammalian oocytes store mRNAs has remained elusive. Cheng et al . discovered that mammalian oocytes, including those in humans, store maternal mRNAs around the mitochondria in a membraneless compartment with hydrogel-like properties. The RNA-binding protein ZAR1 drives the assembly of this compartment, which clusters the mitochondria and protects the mRNAs against degradation. —SMH

Abstract

A membraneless compartment clusters mitochondria and stores maternal mRNAs in oocytes of various mammalian species.

Abstract

INTRODUCTION

Mammalian oocytes accumulate a large number of messenger RNAs (mRNAs) through active transcription as they grow. Transcription ceases during the final stages of oocyte growth and only resumes when the embryonic genome is activated after fertilization. During this period, the oocyte and the embryo can only use the stored mRNAs to synthesize new proteins. Proper storage of maternal mRNAs is thus critical for the maturation of oocytes into fertilizable eggs through meiosis and for early embryonic development after fertilization. However, where and how maternal mRNAs are stored in mammalian oocytes, including human oocytes, has remained elusive.

RATIONALE

RNAs are often stored in membraneless compartments that form by spontaneous phase separation of proteins and/or nucleic acids. Previous studies identified different types of membraneless compartments that store mRNAs in non-mammalian oocytes, such as P granules in Caenorhabditis elegans and Polar granules in Drosophila . We thus set out to identify potential RNA storage compartments in mammalian oocytes.

RESULTS

We analyzed the localization of RNA-binding proteins that were highly expressed in mouse oocytes. We found that the RNA-binding proteins ZAR1, YBX2, DDX6, LSM14B, and 4E-T (EIF4ENIF1) co-localized with mitochondria, forming clusters throughout the cytoplasm. By contrast, they did not co-localize with the Golgi apparatus, recycling endosomes, or lysosomes, and only partially co-localized with the endoplasmic reticulum. Additionally, we stained mRNAs using RNA fluorescence in situ hybridization and found that they were stored in this mitochondria-associated domain. This domain was also present in oocytes of other mammalian species, including humans. Because this domain was distinct from any known RNA-containing compartment, we named it mitochondria-associated ribonucleoprotein domain, or MARDO for short.

MARDO assembly around mitochondria was directed by an increase in mitochondrial membrane potential during oocyte growth. The MARDO gradually appeared as oocytes grew and became most prominent in full-grown oocytes, the mitochondria of which are also the most active. Among the MARDO-localized RNA-binding proteins, ZAR1 played a major role in the assembly of the MARDO. ZAR1, but not other RNA-binding proteins, promoted the coalescence of MARDO foci into hydrogel-like matrices when overexpressed. MARDO coalescence drove the aggregation of mitochondria into giant clusters. Through a series of in vivo and in vitro experiments, we found that the unstructured N-terminal domain of ZAR1 was essential for MARDO assembly and its association with mitochondria. We depleted ZAR1 by gene knockout, RNA interference, and Trim-Away and found that both MARDO formation and mitochondrial clustering were impaired. MARDO formation and mitochondrial clustering were restored by expressing ZAR1 in Zar1 -knockout oocytes. These results confirmed that ZAR1 is essential for MARDO assembly and mitochondrial clustering. Furthermore, live-cell imaging analyses showed that loss of ZAR1 caused severe defects in spindle assembly, chromosome alignment, and cytokinesis during oocyte meiotic maturation.

The MARDO stored translationally repressed mRNAs, some of which are known to become translationally activated during the maturation of oocytes into fertilizable eggs or after fertilization. Loss of ZAR1 not only disrupted the MARDO, but also caused a premature loss of MARDO-localized mRNAs. Maternal mRNAs need to be progressively degraded and replaced by mRNAs transcribed from the embryonic genome to ensure proper embryonic development. The MARDO dissolved during the transition from meiosis I to meiosis II because of proteasomal degradation of ZAR1, which was essential for the timely degradation of maternal mRNAs.

CONCLUSION

In this study, we identified the MARDO, a mitochondria-associated membraneless compartment that stores maternal mRNAs in oocytes of various mammalian species, including humans. Our data reveal how the MARDO coordinates maternal mRNA storage, translation, and degradation to ensure fertility in mammals. The RNA-binding protein ZAR1 promotes MARDO assembly and coalescence into clusters. The MARDO stores translationally repressed mRNAs, some of which are translated during later stages of development. Proteasomal degradation of ZAR1 drives MARDO dissolution in mature eggs to ensure the timely degradation of maternal mRNAs.

Our data also reveal physical and functional interactions between the membraneless MARDO and membrane-bound mitochondria, both of which are maternally contributed compounds that accumulate during oocyte growth.