Phase separation has been found to mediate an important mechanism of protein translational activation during sperm development

In the early morning of August 12, Beijing time, Science published a research paper on the topic of LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs in collaboration with the Liu Mofang Research Group of the Center for Excellence in Molecular and Cell Science (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences and a number of laboratories at home and abroad. The study reported that the RNA-binding protein FXR1 can activate the translation of mRNA in late sperm cells of mice by liquid-liquid phase separation, ensuring the normal progress of the sperm formation process. Activating translation with phase separation, a professor at the French Institute of Human Genetics, published in Science, suggests that although the dynamic changes of RNP particles during development have been described, it is still challenging to analyze how the formation of such particles performs biological functions in the body, and the results have promoted research on this issue.

During sperm cell deformation, as the nucleus is progressively compressed, the transcriptional activity of the genome will gradually decrease until it stops completely. Genes required for late sperm cell development need to be transcribed into messenger ribonucleic acid (mRNA) in advance, then stored in the messenger ribonucleoproteins (mRNPs) in a state of translation inhibition, and then activated for translation at a specific stage of development to synthesize proteins to function. However, scientists know little about how the mRNA stored in these late sperm cells is translated and activated.

In order to explore the translational activation mechanism of mRNA in late sperm cells, the polysome protein profile of mouse testicles at different stages of development was analyzed, and potential translation regulators in late sperm cells were excavated. The results showed that the RNA-binding protein FXR1 was significantly enriched in the polysome component of late sperm cells and was specifically highly expressed in the testes of mice. Further studies found that FXR1 binds to a large group of mRNAs that are specifically translated in late sperm cells and interacts with multiple translation-related factors such as EIF4G3. Germ cell-specific knockout of Fxr1 does not affect the stability of the target mRNA, but results in a decrease in its translational activity and protein expression in late sperm cells. More importantly, Fxr1 knockout mice are azoosperm and male infertile, suggesting that FXR1 is critical for both late sperm cell translation and sperm cell development.

Mechanistic studies have shown that FXR1 has protein concentration-dependent liquid-liquid phase separation (LLPS) properties, and FXR1 and EIF4G3 and other translation-related factors and target mRNA are significantly enriched in FXR1 particles in late sperm cells, suggesting that FXR1 phase separation in late sperm cells may be closely related to its translational activation of target mRNA. Studies have demonstrated at the culture and sperm cell levels using the TRICK reporting system and the lentiviral transduction system that characterize the translation status of intracellular RNA to demonstrate that the ability to separate FXR1 is closely related to its intracellular formation of particles and the translational activation of its target RNA. Using CRISPR-Cas9 combined with hemi-cloning technology, a mouse model of FXR1 mutation with lack of germ cell-specific knock-in phase separation ability was constructed. The mice exhibited azoospermia and male sterility, demonstrating that FXR1 phase separation was necessary for late sperm cell mRNA translation activation and sperm formation.

Based on the research results, the researchers proposed a working model: in spherical sperm cells, the low-level expression of FXR1 can cooperate with other RNA-binding proteins to recognize some newly transcribed mRNAs and assemble into mRNPs in the translation-inhibited state in the cytoplasm; Entering the late stage of sperm cells, a large number of expressed FXR1 undergoes phase transition and forms FXR1 particles, recruits translation machines such as EIF4G3, activates the translation of mRNA stored in FXR1 particles, and ensures sperm cell development and sperm production.

FXR1 particles mediate late sperm cell translation activation

The research work has been funded by the Ministry of Science and Technology, the National Natural Science Foundation of China, the Chinese Academy of Sciences, the Shanghai Municipal Science and Technology Commission, etc., supported by the GTP Center of Molecular Cell Excellence, the Molecular Biology Technology Platform, the Cell Analysis Technology Platform, the Animal Experiment Technology Platform, etc., and assisted by researchers from the University of California, San Diego, Wuhan University, Nanjing Medical University, Shanghai Family Planning Science Research Institute, Baylor Medical College, etc. (Source: Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences)

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