Live blogger: Paul Dylag
Editor: Jennifer Baker
This piece was written live during the 7th annual RNA Symposium, “From Molecules to Medicines,” hosted by the University of Michigan’s Center for RNA Biomedicine. Follow MiSciWriters’ coverage of this event on Twitter with the hashtag #umichrna.
Biomolecular condensates are found throughout plant and animal cells in various organelles that lack membranes, such as the nucleolus and RNA granules. Normally, membraneless organelles would be an issue, as mixing their components with cytoplasm or extracellular fluid may result in mutations. However, there must be some chemical agents that prevent this, as otherwise life would not have evolved to such complex levels. Researchers are still investigating what prevents these issues from occurring, but one category of molecules called pickering agents have been determined to play a key role in this process.
Pickering agents, discovered in 1907, stabilize mixtures of two different liquids that normally do not mix. Pickering agents use nanoscale solid particles that gather between the two previously unmixable layers. The energy required to eject the solid particles is higher than the energy gained from molecules of the same substance joining together. Therefore, separation is unfavorable and the liquids take a longer time to separate.
The action of Pickering agents is similar to adding mustard to vinaigrettes. Without the mustard, the vinaigrette would eventually separate into its separate layers of oil and vinegar, even if thoroughly shaken. The mustard serves as a Pickering agent by coating the oil and preventing its separation from the vinegar, allowing the mixture to remain mixed for a longer time frame. Furthermore, Pickering agents regulate the exchange of molecules between both liquids in response to changes in the environment. These properties of Pickering agents are useful for a variety of industrial applications, including cosmetics, drug innovation, and food science.
Today, Dr. Seydoux is presenting her work on the mechanisms that control molecular exchanges and interactions between P granules, as well as those that determine the total number of P granules in the C. elegans germline. P granules are a class of RNA granules that contain PGL proteins; for example, PGL-3, which is an asymmetrically localized protein and the first protein to localize to P granules. P granules are maternally dependent granules containing RNA and RNA-binding proteins and are known to segregate asymmetrically to give rise to the worm germline. For this talk, she is focusing on two proteins without a three-dimensional structure, named MEG-3 and MEG-4 (MEG stands for maternally expressed gene), that regulate this process. MEG-3 and MEG-4 proteins help assist asymmetric division that give rise to the worm germline. She then explains how MEG-3 and MEG-4 can be classified as cellular Pickering agents because of the way they can form protein clusters that gather on the border of the condensate.
To test her hypothesis, Dr. Seydoux employed the use of live C. elegans embryos to determine 1) the effect of inhibiting MEG proteins on worm development and 2) the motion of fluorescently labeled PGL and MEG proteins that results in the development of location-specific organelles in the embryo. Cell culture was used to determine how MEG proteins affect the internal and external dynamics of the P granules, which would be difficult to measure in live organisms. In experiments where comparisons between cells and worms were possible, they were performed to ensure results were not offset by other elements in live animals, but not in culture. In embryos where MEG-3 and MEG-4 protein synthesis was inhibited, P granule formation was inhibited, resulting in only 70% of mutant embryos being fertile. Thus, P granules enhance species survivability into the next generation, but if a mutation arises it would not endanger the population. In contrast, when MEG-1 and MEG-2 proteins are inhibited, the RNA in the P granules are not able to leave the cytoplasm, yielding 100% sterile embryos. However, more research is needed to determine the full effect that MEG-1 and MEG-2 play in this process.
When imaged under a microscope, P granules appear like little glowing raindrops that are able to conglomerate together via low affinity interactions that depend on the ratio of PGL-3 to MEG-3 and MEG-4 proteins. Limiting the size of P granules allows for increased fertility, as interactions inside smaller P granules and extracellular interactions can be carried out more quickly than in larger P granules due to the smaller surface area to volume ratio. Using fluorescence techniques, Dr. Seydoux found MEG-3 on the surface of PGL-3-containing cells. These MEG-3 proteins move around the granule about 100 times less than PGL-3 proteins. Furthermore, MEG-3 does not inhibit intercondensate interactions within a PGL-3-containing cell, allowing them to form the worm germline, while ensuring the embryo has favorable qualities for reproduction.. It is still unknown whether MEG-4 serves a similar function.
Towards the end of the presentation, Dr. Seydoux emphasized the importance of proper P granule synthesis. The binding site of PGL-3 proteins is specific to the carboxyl terminus of MEG-3 and MEG-4 proteins. Without these, embryo viability is decreased. She also mentioned asymmetric break coding is upstream of P granules. Since P granules respond to the asymmetric break, if asymmetric division is delayed or eliminated, the P granules would not localize and would end up degraded.
What are the next steps?
There are many questions about the functions and regulations of MEG proteins remaining. Among her list of future directions, Dr. Seydoux is especially interested in answering the follow questions:
- How are the MEG-3 proteins localized – what gradient controls their movement?
- What role do MEG-1 and MEG-2 proteins play in the P granules of germline lineages?
- Are MEG proteins present in the same concentrations in P granules of adult C. elegans?
Dr. Geraldine Seydoux is a Professor of Molecular Biology and Genetics at the Johns Hopkins University School of Medicine. She obtained her Ph.D. in 1991 from Princeton University. After obtaining her Ph.D., Dr. Seydoux did postdoctoral training at the Carnegie Institution of Washington with Andy Fire. In 1995, she joined the faculty at Johns Hopkins University School of Medicine. She is an investigator of the Howard Hughes Medical Institute and a member of the National Academy of Sciences and of the European Molecular Biology Organization (EMBO). Dr. Seydoux’s research focuses on germline development. The Seydoux lab has developed methods for genome editing that take advantage of a highly efficient gene conversion mechanism in the germline and identified intrinsically-disordered proteins that stabilize RNA granules in the cytoplasm by functioning as surface-tension reducing agents. In 2022, Dr. Seydoux was awarded the Gruber Prize in Genetics alongside Lehmann and Priess for her work in detailing how sex cells transmit genetic information across generations in Caenorhabditis elegans.