LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs

Postmeiotic spermatids use a unique strategy to coordinate gene expression with morphological transformation, in which transcription and translation take place at separate developmental stages, but how mRNAs stored as translationally inert messenger ribonucleoproteins in developing spermatids become activated remains largely unknown. Here, we report that the RNA binding protein FXR1, a member of the fragile X–related (FXR) family, is highly expressed in late spermatids and undergoes liquid-liquid phase separation (LLPS) to merge messenger ribonucleoprotein granules with the translation machinery to convert stored mRNAs into a translationally activated state. Germline-specific Fxr1 ablation in mice impaired the translation of target mRNAs and caused defective spermatid development and male infertility, and a phase separation–deficient FXR1L351P mutation in Fxr1 knock-in mice produced the same developmental defect. These findings uncover a mechanism for translational reprogramming with LLPS as a key driver in spermiogenesis. Description Driving translation in spermatids Translation control becomes a key mechanism for gene regulation during postmeiotic male germ cell differentiation because of the uncoupling between transcription and translation. Kang et al. identified a member of the fragile X-related (FXR) protein family, FXR1, as a translation activator for stored mRNAs to instruct spermiogenesis in mice (see the Perspective by Ramat and Simonelig). Mechanistically, highly elevated FXR1 in late spermatids forms condensates through phase separation to organize the stored mRNAs into FXR1 granules, and then recruits the translational machinery to activate translation. Either germline-specific depletion of Fxr1 or modeling a phase separation–deficient mutation in Fxr1 similarly causes spermatogenic failure and male infertility in mice. —DJ Phase separation of the RNA-binding protein FXR1 links translation activation to spermatid development in mice. INTRODUCTION In mammals, spermiogenesis (postmeiotic male germ cell differentiation) is a highly orchestrated developmental process controlled by a group of genes collectively referred to as spermiogenic genes. Because nuclear condensation during spermiogenesis gradually halts transcription, spermiogenic genes are transcribed in advance during the earlier stages of male germ development and stored as translationally inert messenger ribonucleoproteins (mRNPs) in developing spermatids until they are needed for translation. Such inert mRNPs are usually organized into mRNP granules called germ granules, which serve as storage facilities for nontranslating mRNAs in various types of germ cells. However, little is known about how those mRNAs stored in inert mRNPs are activated during late spermiogenesis. RATIONALE To understand how translationally inert mRNAs are activated during spermiogenesis, we screened potential translational regulators by proteomic analysis of polysomes from mouse testes. FXR1, a member of the fragile X–related (FXR) protein family, stood out from the screen as a translational regulator in late spermatids. By performing eCLIP and polysome profiling, in combination with generating a germline-specific Fxr1 knockout (Fxr1cko) mouse model, we investigated whether FXR1 is required for translation activation in late spermatids. To decipher the mechanism underlying FXR1-mediated translation regulation, we identified the potential cofactor(s) of FXR1 in mouse testes using immunoprecipitation coupled with mass spectrometry. We observed the formation of FXR1 granules through liquid-liquid phase separation (LLPS), which recruits translation factors in late spermatids, and used the TRICK (translating RNA imaging by coat protein knock-off) reporter system to determine whether FXR1 LLPS is required for target translation in cultured cells. To further investigate whether FXR1 LLPS is critical for target translation in mouse spermatids, we ectopically expressed wild-type FXR1, LLPS-deficient FXR1L351P mutants, or LLPS-restored FXR1L351P-IDRFUS mutants in Fxr1cko testes using lentiviral testis transduction. Finally, by generating germline-specific Fxr1L351P knock-in mice, we determined whether FXR1 LLPS is indispensable to translation activation in late spermatids, spermiogenesis, and male fertility in mice. RESULTS We found that FXR1 was much more enriched in polysomes from 35-day postpartum (dpp) testes relative to 25-dpp testes, suggesting a role for FXR1 in translation activation in late spermatids. We identified a group of 770 mRNAs as being likely direct FXR1-activated targets, and demonstrated that germline-specific Fxr1 deletion in mice markedly reduced target translation in late spermatids. Consistent with FXR1 functioning in translation activation in late spermatids, Fxr1cko male mice were infertile and displayed spermatogenic failure at late spermiogenesis. Interestingly, we observed a pronounced up-regulation of FXR1 and the formation of abundant, distinct condensates in late spermatids, suggesting concentration-dependent LLPS. Mechanistic studies revealed that FXR1 undergoes LLPS to form condensates that assemble target mRNAs as mRNP granules and then recruit translational machinery to activate the stored mRNAs. Consistently, ectopic expression of wild-type FXR1 or FXR1L351P-IDRFUS, but not FXR1L351P, activated target translation in cultured cells and successfully rescued target translation in late spermatids and spermiogenesis in Fxr1cko mice. Furthermore, Fxr1L351P knock-in mutant mice highly phenocopy Fxr1cko mice, directly supporting the indispensability of FXR1 LLPS to target translation in late spermatids, spermiogenesis, and male fertility in mice. CONCLUSION Our findings demonstrate that FXR1 is an essential translation activator that instructs spermiogenesis in mice and unveil a key contribution of FXR1 LLPS to the translation activation of stored mRNAs in mouse spermatid and male fertility in mice. In addition, our study pinpoints the importance of LLPS in a developmental process in vivo. FXR1-containing granules mediate translation activation in late spermatids. During late spermiogenesis, elevated FXR1 undergoes LLPS to assemble target mRNAs as FXR1 mRNP granules that recruit translational machinery by interacting with the eukaryotic translation initiation factor 4 gamma 3 (EIF4G3) to activate the stored mRNAs in late spermatids. These phase-separated FXR1 granules drive a large translation program to instruct spermatid development and sperm production in mice.