One emerging paradigm of cellular organization of RNA and RNA binding proteins is the formation of membraneless organelles (MLOs). Examples of MLOs include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and post-translational 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 C. elegans can cause an inadvertent stress and trigger phase transitions of RNA binding proteins. We show this imaging-associated stress stimulates decondensation of multiple germ granule proteins, and condensation of several P-body proteins. Proteins within larger RNP 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 RNP granules of arrested oocytes may have distinct phases which we characterize in our companion paper.

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 C. elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In C. 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 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 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.

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 C. elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In C. elegans, when oogenesis undergoes an 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 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 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.