The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis to maintain translational repression of maternal mRNA and oocyte quality.

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo C. elegans oogenesis model to determine the intrinsic properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and the Lsm protein, CAR-1. We demonstrate that MEX-3 undergoes liquid-liquid phase separation and appears to have intrinsic gel-like properties in vitro . We also identify novel roles for the CCT chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. CCT and actin also oppose the expansion of ER sheets that may promote ectopic condensation of RNA-binding proteins that are associated with de-repression of maternal mRNA. This regulatory network is essential to preserve oocyte quality, prevent infertility, and may have implications for understanding the role of hMex3 phase transitions in cancer. Significance statement: The molecular mechanisms that regulate phase transitions of oogenic RNA-binding proteins are critical to elucidate but are not fully understood.We identify novel regulators of RNA-binding protein phase transitions in maturing oocytes that are required to maintain translational repression of maternal mRNAs and oocyte quality.This study is the first to elucidate a regulatory network involving the CCT chaperonin, actin, and the ER for phase transitions of RNA-binding proteins during oogenesis. Our findings for the conserved MEX-3 protein may also be applicable to better understanding the role of hMex3 phase transitions in cancer.

Large RNP granules in Caenorhabditis elegans oocytes have distinct phases of RNA-binding proteins.

The germ line provides an excellent in vivo system to study the regulation and function of RNP granules. Germ granules are conserved germ line-specific RNP granules that are positioned in the Caenorhabditis elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In Caenorhabditis elegans, when oogenesis undergoes extended meiotic arrest, germ granule proteins and other RNA-binding proteins assemble into much larger RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. To gain insight into the function of oocyte RNP granules, in this report, we characterize distinct phases for four protein components of RNP granules in arrested oocytes. We find that the RNA-binding protein PGL-1 is dynamic and has liquid-like properties, while the intrinsically disordered protein MEG-3 has gel-like properties, similar to the properties of the two proteins in small germ granules of embryos. We find that MEX-3 exhibits several gel-like properties but is more dynamic than MEG-3, while CGH-1 is dynamic but does not consistently exhibit liquid-like characteristics and may be an intermediate phase within RNP granules. These distinct phases of RNA-binding proteins correspond to, and may underlie, differential responses to stress. Interestingly, in oocyte RNP granules, MEG-3 is not required for the condensation of PGL-1 or other RNA-binding proteins, which differs from the role of MEG-3 in small, embryonic germ granules. Lastly, we show that the PUF-5 translational repressor appears to promote MEX-3 and MEG-3 condensation into large RNP granules; however, this role may be associated with regulation of oogenesis.

Imaging-associated stress causes divergent phase transitions of RNA-binding proteins in the Caenorhabditis elegans germ line.

One emerging paradigm of cellular organization of RNA and RNA-binding proteins is the formation of membraneless organelles. Examples of membraneless organelles include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and posttranslational modifications, as well as extracellular stresses, can stimulate the condensation of proteins into granules. For example, the assembly of stress granules induced by oxidative stress, osmotic stress, and heat stress has been well characterized in a variety of somatic cell types. In the germ line, similar stress-induced condensation of proteins occurs; however, less is known about the role of phase separation during gamete production. Researchers who study phase transitions often make use of fluorescent reporters to study the dynamics of RNA-binding proteins during live cell imaging. In this report, we demonstrate that common conditions of live-imaging Caenorhabditis elegans can cause an inadvertent stress and trigger phase transitions of RNA-binding proteins. We show that this imaging-associated stress stimulates decondensation of multiple germ granule proteins and condensation of several P-body proteins. Proteins within larger ribonucleoprotein granules in meiotically arrested oocytes do not appear to be as sensitive to the stress as proteins in diakinesis oocytes of young hermaphrodites, with the exception of the germ granule protein PGL-1. Our results have important methodological implications for all researchers using live-cell imaging techniques. The data also suggest that the RNA-binding proteins within large ribonucleoprotein granules of arrested oocytes may have distinct phases, which we characterize in our companion article.

An Emerging Role for Post-translational Modifications in Regulating RNP Condensates in the Germ Line.

RNA-binding proteins undergo regulated phase transitions in an array of cell types. The phase separation of RNA-binding proteins, and subsequent formation of RNP condensates or granules, occurs during physiological conditions and can also be induced by stress. Some RNP granules have roles in post-transcriptionally regulating mRNAs, and mutations that prevent the condensation of RNA-binding proteins can reduce an organism's fitness. The reversible and multivalent interactions among RNP granule components can result in RNP complexes that transition among diffuse and condensed states, the latter of which can be pathological; for example, in neurons solid RNP aggregates contribute to disease states such as amyotrophic lateral sclerosis (ALS), and the dysregulation of RNP granules in human germ cells may be involved in Fragile X-associated primary ovarian insufficiency. Thus, regulating the assembly of mRNAs and RNA-binding proteins into discrete granules appears to provide important functions at both cellular and physiological levels. Here we review our current understanding of the role of post-translational modifications (PTMs) in regulating the condensation of RNA-binding proteins in the germ line. We compare and contrast the in vitro evidence that methylation inhibits phase separation of RNA binding proteins, with the extent to which these results apply to the in vivo germ line environment of several model systems. We also focus on the role of phosphorylation in modulating the dynamics of RNP granules in the germ line. Finally, we consider the gaps that exist in our understanding of the role of PTMs in regulating germ line RNP granules.