In:
Science, American Association for the Advancement of Science (AAAS), Vol. 377, No. 6607 ( 2022-08-12)
Abstract:
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 ( Fxr1 cko ) 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 FXR1 L351P mutants, or LLPS-restored FXR1 L351P -IDR FUS mutants in Fxr1 cko testes using lentiviral testis transduction. Finally, by generating germline-specific Fxr1 L351P 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, Fxr1 cko 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 FXR1 L351P -IDR FUS , but not FXR1 L351P , activated target translation in cultured cells and successfully rescued target translation in late spermatids and spermiogenesis in Fxr1 cko mice. Furthermore, Fxr1 L351P knock-in mutant mice highly phenocopy Fxr1 cko 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.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abj6647
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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