Loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction in mouse spermatogonia

Loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction in mouse spermatogonia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction in mouse spermatogonia Yuxuan He, Rong Gan, Jiahao Zhang, Weitao Dong, Dong Zhang, Xiaoyun Pang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9099620/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Ribosomal proteins (RPs) regulate p53 activation, which is mediated by mouse double minute 2 homolog (MDM2), ribosomal protein L5 (RPL5) and/or ribosomal protein L11. Thus, this study aimed to explore whether ribosomal protein L27a (RPL27a) interacts with RPL5 and regulates p53 activation in mouse spermatogonia. Immunohistochemistry and immunofluorescence were used to analyze protein expression in vivo . Protein and mRNA expression was analyzed by immunoblotting and real-time polymerase chain reaction, respectively. The bindings of RPL27a with RPL5, and MDM2 were verified by molecular docking and glutathione S-transferase (GST)-fusion protein assays. Co-immunoprecipitation and in vitro ubiquitination assays were used to confirm protein interactions and p53 accumulation. An adeno-associated virus was used to knock down RPL27a in vivo . The results showed that knockdown of RPL27a weakened the binding of RPL27a to RPL5 and MDM2, but promoted the interaction between RPL5 and MDM2. Meanwhile, knockdown of RPL27a induced p53-dependent cell cycle arrest and RPL5-dependent p53 activation in mouse spermatogonial GC-1 spg cells. Moreover, RPL27a knockdown induced apoptosis of spermatogonia via the accumulation of p53, as verified in vivo . This study indicates that RPL27a negatively regulates p53 activation via enhancing the RPL5-MDM2 interaction in spermatogonia, and provides a better understanding of male infertility induced apoptosis of spermatogonia. Apoptosis Protein interaction Ribosomal stress Ribosomal protein L27a Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Spermatogonia as an initial status of spermatogenic cells plays an important role in maintaining spermatogenesis [ 1 ]; however, excessive apoptosis of spermatogonia disrupts normal spermatogenesis, thereby resulting in inadequate sperm counts and quality defects [ 2 ], which is one cause of male infertility [ 3 ]. Infertility involves complex pathogenetic mechanisms [ 4 ], and is affected about 15% of all couples worldwide [ 5 ]. Thus, revealing the mechanisms of spermatogonial apoptosis can help understand the process of male infertility. The protein p53 is crucial in DNA damage repair, cell cycle arrest and apoptosis [ 6 ], is thought to be critical in regulating the apoptosis of spermatogonia [ 7 ]. Normal ribosome biogenesis is necessary for maintaining cell homeostasis [ 8 ], but any interference with this complex process can cause ribosomal stress that leads to p53 activation through the interaction between ribosomal proteins (RPs) and MDM2 [ 9 ]. A variety of RPs interact with MDM2 and form a RP–MDM2-p53 multi-complex, which provides a potential link between ribosomal stress and p53 activation [ 10 ]. The binding of RPL26 [ 11 ] and RPL23 [ 12 ] with MDM2 can be enhanced under ribosomal stress, thereby activating p53. RPL5 and RPL11 are effectors of ribosomal stress that, together with MDM2, compose the RPL5/RPL11-MDM2-p53 complex [ 13 ]. Downregulation of RPS19 [ 14 ], RPL23a [ 15 ] and RPS27a [ 16 ] has been verified to increase p53 activity in an RPL5- or RPL11-dependent manner. RPL27a is a ribosomal protein constituting the 60S large ribosomal subunit [ 17 ]. Previous studies have shown that the deletion of RPL27a causes p53 accumulation and activation through MDM2–p53 [ 18 ]. We have screened RPL27a as a linker between DNA damage and apoptosis of spermatogonia, and we have proposed that loss of RPL27a expression affects p53 accumulation through the RPL5-MDM2-p53 complex [ 19 ]. However, RPL27a regulates p53 accumulation whether it is involved in RPL5–dependent that is still unclear. Thus, the relationship of RPL27a expression and p53 activation in apoptosis of spermatogonia was further investigated in the present study. The interactions of RPL27a and RPL5, of RPL27a and MDM2 were confirmed, which inhibits MDM2-mediated p53 ubiquitination in spermatogonia. Therefore, balanced levels of RPL27a are crucial for maintaining normal levels of p53, and the loss of RPL27a expression enhances the binding of RPL5 and MDM2, thus leading to activation of p53 in spermatogonia. These results provide insight into the relationship of RPs and p53 activation in apoptosis of spermatogonia. Materials and methods Animal Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou, China) provided the mice for irradiation, and the Obio Biotech (Shanghai, China) provided the mice for AAV experiments. The male C57BL/6 mice were 4 weeks old with body weight 16–18 g. Terminal dUTP nick end labeling (TUNEL) assay Apoptotic spermatogenic cells were detected with TUNEL assays (Servicebio, Wuhan, China) according to previous study [ 19 ]. Briefly, the TUNEL reaction was finished, then the sections were stained with 4, 6-diamidino-2-phenylindole (DAPI) (Servicebio) for 10 min at 5 µg/mL and covered with anti-fade mounting medium. Cell culture and transient transfection GC-1 spermatogonia cells derived from mouse (no. BNCC338398) (BeNa, Culture Collection, Beijing, China) that were cultured in high-glucose DMEM (Meilunbio, Dalian, China) supplemented with 10% fetal bovine serum (ExCell bio, Shanghai, China) under controlled conditions (37°C, 5% CO 2 ). The cells were harvested 48 h after transfection and then treated with lysis buffer for immunoblotting analysis. Plasmids, drugs, antibodies, and siRNAs His-tagged MDM2 and RPL5 expression plasmids were constructed by inserting at Nco I and Xho I sites. The full-length RPL27a was inserted into the pEGX.6P.1 vector, then constructed the GST-RPL27a bacterial expression vector. The RPL27a cDNA was inserted into the pcDNA3.1-3xFlag-C vector, then constructed the overexpression of Flag-tagged RPL27a plasmids. HA-tagged MDM2 and His-tagged ubiquitin B (Ub) expression plasmids were constructed by inserting the full-length MDM2 cDNA and Ub into pUC57 vector, after double digestion and then cloned into the pIRES2-ZsGreen1 vector. The pEX-3-RPL27a cDNA was amplified using the following mRNA primers: 5′-GCTCTAGATTACTTGTCTTCTGGTTTGT-3′ and 5′-CCCAAGCTTATGCAGATTTTCGTGAAAAC-3′. The Flag-RPL27a, HA-MDM2, and His-RPL5 fragments were also cloned using PCR and inserted into the pIRES2-ZsGreen1 vector. The plasmid informations were listed in Online Resource 1. Lipo2000 (no. 2359258) was obtained from Invitrogen (Carlsbad, CA, USA), and MG132 (no. HY-13259) was obtained from Medchemexpress (NJ, USA). Antibodies to the following proteins were used for immunoblotting analysis: β-actin (no. ab8227), GAPDH (no. ab181602), RPL27a (no. ab74731), MDM2 (no. ab16895), nucleolin (no. ab129200), p53 (no. ab74731), p21 (no. ab109199), ATM (phospho S1981) (no. ab81292), γH2Ax (phospho S139) (no. ab81299), 53BP1 (no. ab175933), Rad51 (no. ab133534) (Abcam, Cambridge, UK), RPL5 (no. YT4117) and RPL27a (no. YT4103) (Immunoway, NJ, USA), B23 (no. sc-271737 FITC) (Santa Cruz, California, USA) and MDM2 (no. GTX100531) (Genetex, CA, USA), Bax (no. AF0120), Bcl2 (no. AF6139) and Cleaved-Caspase3 (no. AF7022) (Affinity, Melbourne, Australia), and Caspase3 (no. 19677-1-AP) (Proteintech, Wuhan, China). β-actin and GAPDH were used as protein controls to normalize the levels of protein expression. The sequences of RPL27a and p53 were knocked down with siRNAs synthesized by Genepharma (Shanghai, China), as described in previous study [ 19 ]. The three different siRNAs to RPL5 were synthesized by Genepharma, as shown in Online Resource 2. Immunoblotting (IB) The protocols of IB was according to our previous study [ 20 ]. Briefly, a total of 20–40 µg protein was subjected to electrophoresis and transferred to polyvinylidene difluoride membranes, then blocked with 5% nonfat milk and incubated overnight with primary antibodies at 4°C. Finally, the bands were detected by immunoreactivity.. Cell cycle and apoptosis analysis The analysis of cell cycle with propidium iodide (PI) and apoptosis with Annexin V/PI assay by flow cytometer (Beckman Coulter, Inc., CA, USA) according to our previous studies [ 16 , 19 ]. Data were analyzed with the ModFit software program. Measurement of the ribosomal subunit ratio GC-1 cells were collected as described in a previous study and extracted on ice with 20 mM HEPES (pH 7.4). The samples were cooled in ice water, added to a 5%–70% sucrose density gradient (20 mM HEPES buffer) and analyzed by the absorbance at 254 nm (Biocomp, CA) [ 21 ]. GST-fusion assay GST-fusion assays were performed according to the protocols [ 22 ]. Briefly, the expression plasmids of His-tagged RPL5 and His-tagged MDM2 were transfected into Escherichia coli BL21 ( E. coli ). Then, the His-RPL5 and His-MDM2 was purified with an Ni 2+ -NTA column (Thermo Fisher Scientific, USA). Finally, the levels of binding proteins were determined by IB with anti-S-Tag and GST antibodies. Co-immunoprecipitation (co-IP) and in vitro ubiquitination assay GC-1 cells were transfected with tagged plasmids, or vector control after transfection with RPL27a-siRNA for 24 h, the in vitro ubiquitination experiments needed to treat with 40 µM MG132 for 6 h. Subsequently, 70% of the lysate was incubated with anti-Flag, anti-HA or anti-His monoclonal antibody (Cell Signaling Technology, USA), or control IgG [ 23 ]. H&E, immunofluorescence, immunohistochemistry and in situ hybridization The deparaffinized and dehydrated testicular sections were stained with hematoxylin solution, then were stained with eosin and sealed with neutral gum. The permeabilized testicular sections and GC-1 cells were incubated with primary antibody (1:100), then with secondary antibody: FITC (GB22303; Servicebio)/Cy3 (GB21401; Servicebio) for 1 h at room temperature. The sections and cells were stained for with DAPI (Servicebio) before covering [ 24 ]. The GC-1 cells were scanned and analyzed with a confocal laser microscope (LSM, Carl Zeiss AG, Germany) or observed under a biomicroscope (BX53, Olympus, Tokyo, Japan). The protocols of immunohistochemistry was described as the previously [ 24 ]. Briefly, the incubation of secondary antibody was finished, then the testicular sections were stained with 3,3´-diaminobenzidine. The in situ hybridization assay was according to previous study [ 25 ]. The deparaffinized and dehydrated testicular sections were incubated with the digoxigenin-labeled RPL27a cDNA probe sequence 5′- TGCTAATCTGTCCAGTATTAAGTGACCAGTCGTCCT-3′ at 42°C for 3 h. Finally, the sections were incubated with IgG secondary antibody labeled Cy3 (GB21401; Servicebio), then stained with DAPI (Servicebio) before covering. Quantitative PCR analyses The primers was designed and synthesized by TaKaRa company (Dalian, China), and the DDCt method was used to analyze the level of mRNAs. The primers used were 5′-ATTACCACTTGAAGAGGAACCAGAG-3′ and 5′-TTGCCCAGAACTTTGTAGTAGCC-3′ (RPL27a); 5′-TGGAGGTGAATGGAGGTGAATAC-3′ and 5′- TCAGAGTCATAACCAGGGAATCG-3′ (RPL5); 5′-AAGTGTGCCGTTGTCTCTTCG-3′ and 5′-AAATCTGTCAGGCTGGTCTGC-3′ (p21); 5′-CCATCTACAAGAAGTCACAGCAC-3′ and 5′-GCCATCACCATCGGAGCAG-3′ (p53); 5′-GGTTGAGGGCCACCTTATTT-3′ and 5′-GAAGAAAGACCGGGAAGAGAAA-3′ (47S pre-rRNA); 5′-GGCAGGGGAGAGTGATACAGA-3′ and 5′-AAGTCAAAGTTCCACCGTTCTCG-3′ (MDM2) [ 26 ]. RPL27a knockdown in vivo An adeno-associated virus (AAV) was used to knock down RPL27a in vivo. The mouse RPL27a cDNA (NM_011975.3) was cloned into AAV expression plasmids with the primers described in Online Resource 3. The recombinant AAV serotype-PHP.eB (AAV-PHP.eB) was produced by transfection of AAV expression plasmids into human embryonic kidney 293 cells. Male mice were used for experiments and subjected to injections as previously reported [ 27 ]. The mice were anaesthetized with isoflurane, and administered 5×10 11 vgs virus in a 300 µl volume by tail-vein injection. The animals were sacrificed 3 weeks post injection, and testes were dissected for analysis. The viruses infecting the seminiferous tubules of the testes were observed and imaged to detect GFP expression in frozen sections (Online Resource 4). Statistics All testicular sections were scanned and analyzed in Panoramic MIDI software (3DHISTECH, Budapest, Hungary). GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The statistical differences between two groups were analyzed by student’s t- test. Multiple groups of statistical analysis were assessed with one-way analysis of variance (ANOVA) with Tukey’s test. Data are indicated as mean ± SD. * p < 0.05, ** p < 0.01 and # p < 0.001 were considered thresholds of statistical significance. Results Knockdown of RPL27a induces p53 activation, promotes apoptosis and cell cycle arrest in GC-1 cells We analyzed the effect of RPL27a knockdown on apoptosis, cell cycle arrest, and expression of RPL5, p53 and MDM2 in GC-1 cells. The levels of RPL5 mRNA by different siRNAs is shown in Online Resource 5. The expression of p53 protein (Fig. 1 A, B) and mRNA (Fig. 1 C), as well as the p53 target genes p21 and MDM2 (Fig. 1 C), increased significantly by knockdown of RPL27a. The fluorescence signals of RPL27a protein were weakened in the nucleoli and cytoplasm (Fig. 1 D), whereas the fluorescence signals of p53 (Fig. 1 E) and MDM2 (Fig. 1 F) were enhanced in the nucleus and cytoplasm. These results indicated that the decreased level of RPL27a resulted in activation of p53 and p53 target genes p21 and MDM2 , and this process was associated with RPL5. The activation of p53 induced apoptosis, whereas the knockdown of RPL27a significantly increased the apoptotic ratio (Fig. 1 G). Consistently with these findings, RPL27a knockdown significantly increased the ratio of cells in G1 phase (Fig. 1 H), thereby indicating that RPL27a knockdown activates p53 and causes cell cycle arrest, which leads to spermtogonial apoptosis. Knockdown of RPL27a alters the ribosomal subunit ratio of GC-1 cells RPL27a knockdown decreased the expression of 47S pre-rRNA (Fig. 1 C), thereby indicating that knockdown of RPL27a inhibited rRNA synthesis. Nucleolin (NCL) [ 28 ] and nucleophosmin (B23) [ 29 ], two nucleolar integrity marker proteins, were selected to determine the effect of RPL27a knockdown on nucleolar integrity. RPL27a knockdown increased the fluorescence signal of NCL (Fig. 1 I) and B23 (Fig. 1 J); thus, the nucleolar structure in GC-1 cells may be damaged by RPL27a knockdown. The 80S:60S and 80S:40S ratio decreased in the RPL27a knockdown cells that analyzed by polysome profiles (Fig. 1 K and 1 L). Therefore, RPL27a knockdown changes the ribosomal subunit ratio and could generate nucleolar stress. RPL27a knockdown-caused cell cycle arrest and apoptosis is p53-dependent, which requires RPL5 GC-1 cells were co-transfected with RPL27a-siRNA and p53-siRNA to determine whether the G1 phase arrest induced by RPL27a knockdown was p53 dependent. The RPL27a knockdown-induced increases in p21 and MDM2 levels (Fig. 2 A) and the p21 mRNA level (Fig. 2 B), which eliminated by co-transfected with p53-siRNA. The representative images of flow cytometry in GC-1 cells by co-transfected with si-p53 and RPL27a were shown in Fig. 2 C. RPL27a knockdown-induced G1 phase arrest (Fig. 2 D) and apoptosis (Fig. 2 E), which eliminated by co-transfected with p53-siRNA. Therefore, RPL27a knockdown-caused cell cycle arrest and apoptosis is p53-dependent. GC-1 cells were co-transfected with RPL27a-siRNA and RPL5-siRNA to determine whether the p53 activation caused by RPL27a knockdown was RPL5 dependent. The RPL27a knockdown-induced increases in p21 and MDM2 levels (Fig. 3 A) and the p21 mRNA level (Fig. 3 B), which eliminated by co-transfected with RPL5-siRNA. The representative images of flow cytometry in GC-1 cells by co-transfected with si-RPL5 and RPL27a were shown in Fig. 3 C. RPL27a knockdown-induced G1 phase arrest (Fig. 3 D) and apoptosis (Fig. 3 E), which eliminated by co-transfected with RPL5-siRNA. Therefore, RPL27a knockdown activates p53 activation in RPL5-dependent manner. Knockdown of RPL27a enhances the RPL5 and MDM2 interaction GST-fusion assays demonstrated that RPL27a co-interacted with MDM2 and RPL5 in vitro . Figure 4 A and 4 B show that purified His-RPL5 and His-MDM2 were bound by purified GST-RPL27a protein but not GST alone. These results demonstrated that RPL27a binds both with RPL5 and MDM2 in GC-1 cells. The interaction of RPL27a with MDM2 and of RPL27a with RPL5 ( Fig. 4 C); lane 6 compared to lane 3 in lower panel) was enhanced. The above results suggested that RPL27a binds RPL5 and MDM2, and exogenous overexpression of RPL27a promotes their interaction. In addition, GC-1 cells were transfected together with HA-MDM2 and RPL27a-siRNA, and the protein interaction and expression were analyzed with co-IP and IB. The interaction of MDM2 with RPL5 ( Fig. 4 D); lane 9 compared to lane 8 in middle panel) was enhanced, but the interaction of MDM2 with RPL27a ( Fig. 4 D); lane 9 compared to lane 8 in lower panels) was weakened by RPL27a knockdown. These results suggested that MDM2 interacts with RPL27a and RPL5; when RPL27a was knocked down, the interaction between RPL5 and MDM2 was promoted. Knockdown of RPL27a stabilizes p53 His-Ub plasmids and RPL27a-siRNA was transfected into GC-1 cells to analyze the effect of RPL27a knockdown on p53 ubiquitination. Figure 4 E shows that RPL27a knockdown partially rescued the p53 accumulation (lane 3 compared to lane 2 in upper panel), and the expression of RPL5, p53 and MDM2 increased after RPL27a knockdown ( Fig. 4 E); lane 3 compared to lane 2). These results suggested that RPL27a knockdown inhibits MDM2-mediated ubiquitination of p53. RPL27a interacts with RPL5 and MDM2, according to the Co-IP assay in vitro HA-MDM2, Flag-RPL27a and His-RPL5 plasmids were constructed and cloned with different fragments to determine the sites responsible for the binding between RPL27a and MDM2, and co-IP assays were performed. Full-length MDM2 (amino acids 1–489, molecular weight is about 55 KDa) bound to full-length RPL27a (amino acids 1–148) (Fig. 5 A), and full-length RPL27a (amino acids 1–148) bound to full-length MDM2 (amino acids 1–489) (Fig. 5 B). Full-length RPL5 (amino acids 1–297) bound to the full-length RPL27a (amino acids 1–148) (Fig. 5 C), and full-length RPL27a (amino acids 1–148) bound to full-length RPL5 (amino acids 1–297) (Fig. 5 D). Together, these results further suggested that RPL27a interacts both with RPL5 and MDM2. RPL27a interacts both with RPL5 and MDM2, on the basis of molecular docking The hydrogen bonding of the binding interface of RPL5-RPL27a and MDM2-RPL27a was analyzed as shown in Fig. 5 E and Fig. 5 F. The specific amino acid information for hydrogen bonding and hydrophobic interaction is shown in Fig. 5 E and Fig. 5 F. The analysis of the interaction of amino acids at the binding interface between MDM2 and RPL27a revealed that hydrogen bonding and electrostatic interaction were important interactions. The results of protein-protein docking further suggested interactions between RPL27a and RPL5, and RPL27a and MDM2. Knockdown of RPL27a promotes p53 accumulation in spermatogonia in vivo AAV was used to knock down RPL27a in vivo for 3 weeks, then the apoptotic spermatogonia, RPL27a, p53, MDM2 and RPL5 levels in spermatogonia in vivo were analyzed. The decrease in relative testis weight was showed in Fig. 6 A and the significant decrease in RPL27a mRNA expression detected by Real-time PCR in the shRPL27a testes in Fig. 6 B. Treatment of AAV with shRPL27a increased the RPL5, p53 and MDM2 levels in testes (Fig. 6 C, D). Histological observations showed vacuolization of the seminiferous tubules, disordered spermatogenic cells, and diminished spermatogenic cell numbers and types in shRPL27a groups compared with control groups (Fig. 6 E). The apoptotic cells were clearly more abundant in the shRPL27a groups than the control groups Fig. 6 F). Moreover, the RPL27a mRNA expression detected by in situ hybridization in Fig. 6 G. The weak fluorescence signal of RPL27a was observed in spermatogonia with knockdown of RPL27a of testis (Fig. 7 A). In addition, relatively strong staining of p53 (Fig. 7 B), MDM2 (Fig. 7 C) and RPL5 (Fig. 7 D) was observed in spermatogonia with knockdown of RPL27a of testis. These results indicated that RPL27a knockdown induced apoptosis of spermatogonia via the accumulation of p53 through RPL5-MDM2-p53 pathway in vivo. Discussion In this study, RPL27a knockdown induced an increase in NCL and B23, thereby indicating generation of ribosomal stress consistent with IR-induced movement of NCL into the nucleoplasm, and decreased RPL27a expression in the nucleoli and cytoplasm [ 19 ]. In addition, real-time PCR indicated that knockdown of RPL27a decreased the expression of 47S pre-rRNA, thereby inhibiting rRNA synthesis. Therefore, RPL27a knockdown might trigger ribosomal stress, thereby leading to p53 activation. A variety of RPs bind MDM2 and form RP-MDM2-p53 complexes, thus, providing a potential link between ribosomal stress and activation of p53 [ 30 ]. Their decreased expression levels increase p53 activity in a manner dependent on RPL5 or RPL11 [ 31 ]. Because RPL5 and RPL11 are nucleolar stress effectors and sensors, the RPL5/RPL11-MDM2-p53 complex is the classical model of RP and p53 binding [ 32 ]. In this study, co-knockdown of p53 eliminated cell cycle arrest and apoptosis caused by RPL27a knockdown, thus, indicating that the cell cycle arrest and apoptosis caused by RPL27a knockdown was p53 dependent. Moreover, the knockdown of RPL27a caused p53 accumulation in a manner that was RPL5 dependent, because co-knockdown with RPL5 eliminated p53 activation, and attenuated the cell cycle arrest and apoptosis induced by RPL27a knockdown. In this study, RPL27a binds both to MDM2 and RPL5 was verified in spermatogonia by GST assay. In addition, in vitro ubiquitination showed that the knockdown of RPL27a significantly inhibited MDM2-mediated p53 ubiquitination and led to p53 activation; consequently, RPL27a is crucial in negatively regulating p53 activation in spermatogonia. Furthermore, Co-IP experiments were used to further confirm the binding domain that RPL27a bound RPL5 and MDM2, although which amino acid sequences bound each other was unclear, possibly because the linear binding experiment could not fully simulate the true spatial interaction between proteins. However, the protein-protein docking results indicated that both RPL5 and MDM2 formed a stable complex structure with RPL27a. Finally, we confirmed that the knockdown of RPL27a promoted the binding of MDM2 and RPL5, and inhibited MDM2-mediated p53 ubiquitination. The RPL27a knockdown by AAV assay further clarified the promotion of spermatogonia apoptosis via the accumulation of p53 in vivo. Conclusion In conclusion, this study provides further confirmation that RPL27a is a novel regulator of the RPL5-MDM2-p53 complex, and indicate that the loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction and inducing cell cycle arrest and apoptosis of spermatogonia. Our results not only confirmed the binding between RPL27a and the RPL5-MDM2 interaction, but also revealed RPL27a as a novel regulator of the RPL5-MDM2-p53 pathway. Our findings provide a better understanding of the relationship of RPs and p53 activation in the apoptosis of spermatogonia. Abbreviations 4, 6-diamidino-2-phenylindole (DAPI); Ribosomal proteins (RPs); Phosphate buffered saline (PBS); polyvinylidene difluoride membrane (PVDF); Real-time quantitative polymerase chain reaction (PCR). Declarations Acknowledgements This research was partially funded by the Youth Talent Program of “Fuxi” (Gaufx-03Y02), the National Natural Science Foundation of China (11875061, 31902339), the Scientific Research Start-up Funds for Openly-Recruited Doctors of Gansu Agricultural University (2017RCZX-13), the Talent Project of Longyuan (Gansu province), the fund of College of Veterinary Medicine of Gansu Agricultural University (2025DY-01), Youth Innovation and Entrepreneurship (E339221SR0), the Foundation for Youth Doctor of Gansu Province (2021QB-026), the Special Funds for Discipline Construction of Gansu Agricultural University (GAU-XKJS-2018-067). Funding This research was partially funded by the National Natural Science Foundation of China (11875061, 12005042, 31902339), the Youth Talent Program of “Fuxi” (Gaufx-03Y02), the Scientific Research Start-up Funds for Openly-Recruited Doctors of Gansu Agricultural University (2017RCZX-13), the Talent Project of Longyuan (Gansu province) Youth Innovation and Entrepreneurship (E339221SR0), the Foundation for Youth Doctor of Gansu Province (2021QB-026), the Special Funds for Discipline Construction of Gansu Agricultural University (GAU-XKJS-2018-067). Competing interest The authors have no conflict of interest to declare. All authors read and approved the final manuscript Ethics statement The management and euthanasia of laboratory animals complied with the Administration of Affairs Concerning Experimental Animals of the Ministry of Science and Technology of China (1988.11.14), and carried out in accordance with UK Home Office regulations (UK Animals Scientific Procedures Act of 1986; project license PPL PD8CBD97C). Animal experiments were approved by the Ethics Committee and Institutional Animal Care and Use Committee (IACUC) of the Institute of Modern Physics, Chinese Academy of Sciences. Data Availability Statement All related data not included in the manuscript will be available upon request. Author contributions Yuxuan He : Investigation, Methodology, Formal analysis, Validation, Visualization, Software, Data curation, Writing—original draft, Writing—review and editing. Rong Gan : Investigation, Software, Validation, Visualization, Writing—original draft. Jiahao Zhang : Investigation, Methodology, Validation, Visualization. Weitao Dong : Software, Validation, Visualization. Dong Zhang and Xiaoyun Pang : Investigation, Methodology, Resources. Yanyan Wang and Li Xue 7: Investigation, Validation. Jincheng Yang and Rui Luo : Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Validation, Visualization, Writing—original draft, Writing—review and editing. Rong Li : Resources, Validation, Supervision. Xianghong Du : Conceptualization, Funding acquisition, Supervision, Project administration, Writing—review and editing. Hongyan Li : Conceptualization, Methodology, Supervision, Project administration, Writing—review and editing. All authors read and approved the final manuscript. Consent to participate Not applicable. Consent to publish Not applicable. References Cao J, Lin ZB, Tong MH, Zhang YL, Li YP et al (2022) Mechanistic target of rapamycin kinase (Mtor) is required for spermatogonial proliferation and differentiation in mice. Asian J Androl 22:169–176. https://doi.org/10.4103/aja.aja_14_19 Bejarano I, Rodríguez AB, Pariente JA (2018) Apoptosis is a demanding selective tool during the development of fetal male germ cells. Front Cell Dev Biol 6:65. https://doi.org/10.3389/fcell.2018.00065 Marjault HB, Allemand I (2016) Consequences of irradiation on adult spermatogenesis: Between infertility and hereditary risk. Mutat Res Rev Mutat Res 770(Pt B 340–348. https://doi.org/10.1016/j.mrrev.2016.07.004 Prefumo F, Rossi AC (2018) Endometriosis, endometrioma, and ART results: Current understanding and recommended practices. Best Pract Res Clin Obstet Gynaecol 51:34–40. https://doi.org/10.1016/j.bpobgyn.2018.01.09 Yang C, Yao C, Ji Z, Zhao L, Chen H, Li P et al (2021) RNA-binding protein ELAVL2 plays post-transcriptional roles in the regulation of spermatogonia proliferation and apoptosis. Cell Prolif 54:e13098. https://doi.org/10.1111/cpr.13098 Hafner A, Bulyk ML, Jambhekar A, Lahav G (2019) The multiple mechanisms that regulate p53 activity and cell fate. Nat Rev Mol Cell Biol 20:199–210. https://doi.org/10.1038/s41580-019-0110-x de Morais MP, Curado RF, Silva KSE, Moura KK, Arruda JT (2016) Male idiopathic infertility and the TP53 polymorphism in codon 72. Genet Mol Res 15. https://doi.org/10.4238/gmr15048882 Zhao PY, Yao RQ, Zhang ZC, Zhu SY, Li YX, Ren C et al (2022) Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases. Int J Biol Sci 18:2497–2514. https://doi:10.7150/ijbs.70955 Yang K, Wang M, Zhao Y, Sun X, Yang Y, Li X et al (2016) A redox mechanism underlying nucleolar stress sensing by nucleophosmin. Nat Commun 7:13599. https://doi.org/10.1038/ncomms13599 Liu Y, Deisenroth C, Zhang Y (2016) RP-MDM2-p53 pathway: Linking ribosomal biogenesis and tumor surveillance. Trends Cancer 2:191–204. https://doi.org/10.1016/j.trecan.2016.03.002 Zhang Y, Wang J, Yuan Y, Zhang W, Guan W, Wu Z et al (2010) Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26. Nucleic Acids Res 38:6544–6554. https://doi.org/10.1093/nar/gkq536 Watanabe S, Fujiyama H, Takafuji T, Kayama K, Matsumoto M, Nakayama KI et al (2018) GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system. J Cell Sci 131:jcs213009. https://doi.org/10.1242/jcs.213009 Bursać S, Brdovčak MC, Pfannkuchen M, Orsolić I, Golomb L, Zhu Y et al (2012) Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress. Proc Natl Acad Sci U S A 109:20467–20472. https://doi.org/10.1073/pnas.1218535109 Jia Q, Zhang Q, Zhang Z, Wang Y, Zhang W, Zhou Y et al (2013) Transcriptome analysis of the zebrafish model of Diamond-Blackfan anemia from RPS19 deficiency via p53-dependent and -independent pathways. PLoS ONE 8:e71782. https://doi.org/10.1371/journal.pone.0071782 He YX, Guo YX, Zhang Y, Hu JJ, Dong WT, Du XH et al (2021) Ionizing radiation-induced RPL23a reduction regulates apoptosis via RPL11-MDM2-p53 pathway in mouse spermatogonia. Biomed Environ Sci 34:789–802. https://doi.org/10.3967/bes2021.108 Li H, Zhang H, Huang G, Bing Z, Xu D, Liu J et al (2022) Loss of RPS27a expression regulates the cell cycle, apoptosis, and proliferation via the RPL11-MDM2-p53 pathway in lung adenocarcinoma cells. J Exp Clin Cancer Res 41:33. https://doi.org/10.1186/s13046-021-02230-z Terzian T, Dumble M, Arbab F, Thaller C, Donehower LA, Lozano G et al (2011) Rpl27a mutation in the sooty foot ataxia mouse phenocopies high p53 mouse models. J Pathol 224:540–552. https://doi.org/10.1002/path.2891 Alkhatabi HA, McLornan DP, Kulasekararaj AG, Malik F, Seidl T, Darling D et al (2016) RPL27A is a target of miR-595 and may contribute to the myelodysplastic phenotype through ribosomal dysgenesis. Oncotarget 7:47875–47890. https://doi.org/10.18632/oncotarget.10293 Li H, Zhang H, Huang G, Dou Z, Xie Y, Si J et al (2020) Heavy ion radiation-induced DNA damage mediates apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway in mouse spermatogonia. Ecotoxicol Environ Saf 201:110831. https://doi.org/10.1016/j.ecoenv.2020.110831 Li H, He Y, Yan J, Zhao Q, Di C, Zhang H (2016) Comparative proteomics reveals the underlying toxicological mechanism of low sperm motility induced by iron ion radiation in mice. Reprod Toxicol 65:148–158. https://doi.org/10.1016/j.reprotox.2016.07.014 Wong CC, Traynor D, Basse N, Kay RR, Warren AJ (201) Defective ribosome assembly in Shwachman-Diamond syndrome. Blood 118:4305–43122. https://doi.org/10.1182/blood-2011-06-353938 Zhou X, Hao Q, Liao J, Zhang Q, Lu H (2013) Ribosomal protein S14 unties the MDM2-p53 loop upon ribosomal stress. Oncogene 32:388–396. https://doi.org/10.1038/onc.2012.63 Liu Q, Zhang S, Sun Z, Guo X, Zhou H (2019) E3 ubiquitin ligase Nedd4 is a key negative regulator for non-canonical inflammasome activation. Cell Death Differ 26:2386–2399. https://doi.org/10.1038/s41418-019-0308-7 Liu C, Wei J, Xu K, Sun X, Zhang H, Xiong C (2019) CSE1L participates in regulating cell mitosis in human seminoma. Cell Prolif 52:e12549. https://doi.org/10.1111/cpr.12549 Li M, Yu M, Liu C, Zhu H, He X, Peng S et al (2013) miR-34c works downstream of p53 leading to dairy goat male germline stem-cell (mGSCs) apoptosis. Cell Prolif 46:223–231. https://doi.org/10.1111/cpr.12013 Cui D, Li L, Lou H, Sun H, Ngai SM, Shao G et al (2014) The ribosomal protein S26 regulates p53 activity in response to DNA damage. Oncogene 33:2225–2235. https://doi.org/10.1038/onc.2013.170 Rajasekaran S, Thatte J, Periasamy J, Javali A, Jayaram M, Sen D et al (2018) Infectivity of adeno-associated virus serotypes in mouse testis. BMC Biotechnol 18:70. https://doi.org/10.1186/s12896-018-0479-1 Westdorp KN, Sand A, Moorman NJ, Terhune SS (2017) Cytomegalovirus late protein pUL31 alters pre-rRNA expression and nuclear organization during infection. J Virol 91:e00593–e00517. https://doi.org/10.1128/JVI.00593-17 Avitabile D, Bailey B, Cottage CT, Sundararaman B, Joyo A, McGregor M et al (2011) Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin. Proc Natl Acad Sci U S A 108:6145–6150. https://doi.org/10.1073/pnas.1017935108 Zhu Y, Poyurovsky MV, Li Y, Stahl J, Jacq X, Prives C (2009) Ribosomal protein S7 is both a regulator and a substrate of MDM2. Mol Cell 35:316–326. https://doi.org/10.1016/j.molcel.2009.07.014 Xiong X, Zhao Y, He H, Sun Y (2011) Ribosomal protein S27-like and S27 interplay with p53-MDM2 axis as a target, a substrate and a regulator. Oncogene 30:1798–1811. https://doi.org/10.1038/onc.2010.569 Holmberg Olausson K, Nistér M, Lindström MS (2012) p53-dependent and -independent nucleolar stress responses. Cells 1:774–798. https://doi.org/10.3390/cells1040774 Supplementary Files Supplementarymaterials.docx ESM1.doc ESM2.xls ESM3.doc ESM4.doc ESM5.xls floatimage1.png Graphical Abstract Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 26 Apr, 2026 Reviewers invited by journal 30 Mar, 2026 Editor assigned by journal 18 Mar, 2026 First submitted to journal 16 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9099620","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614740699,"identity":"deed0d25-9ec7-4a31-b977-38990787406e","order_by":0,"name":"Yuxuan He","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"He","suffix":""},{"id":614740700,"identity":"45a0b3c7-be82-453f-9e89-a2670d2d822d","order_by":1,"name":"Rong Gan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDADNvbGxocfSNLCx3O42ViCJC1yEultAjzEqJR3P3vsw8cdtQxskg/bGCQY7OR0GwhoMTyTlzxz5pnjDGzSiW0PChiSjc0OENLSkGPMzNt2DKSl3UCC4UDiNoJa+t9AtUgebJPgIUaLvATYlhoGNglGIrUYSLwxZpzZdoCBjScRGMgGRPhFvj/HmOFjWx2DfPvxhw8/VNjJEdRiAFFwuL4BwiWgHGwLRGkdEUpHwSgYBaNgxAIAo388nn1qqxAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0006-1761-5310","institution":"Institute of Modern Physics Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Rong","middleName":"","lastName":"Gan","suffix":""},{"id":614740701,"identity":"c63e854d-d94a-4f0e-9c49-90dbea751eba","order_by":2,"name":"Jiahao Zhang","email":"","orcid":"","institution":"Gansu Agricultural 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University","correspondingAuthor":false,"prefix":"","firstName":"Jincheng","middleName":"","lastName":"Yang","suffix":""},{"id":614740708,"identity":"cfafd90b-02d1-480c-9139-2281724bad13","order_by":9,"name":"Rui Luo","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Luo","suffix":""},{"id":614740709,"identity":"44b43a1f-2cc9-4741-997b-3cad9c2eda7a","order_by":10,"name":"Rong Li","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Li","suffix":""},{"id":614740710,"identity":"c515276a-94eb-4a89-88dd-e45b1dcb6beb","order_by":11,"name":"Xianghong Du","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xianghong","middleName":"","lastName":"Du","suffix":""},{"id":614740711,"identity":"dd46ab07-5756-4d1f-8b62-0725e01c0ee4","order_by":12,"name":"Hongyan Li","email":"","orcid":"https://orcid.org/0000-0002-9424-8247","institution":"Institute of Modern Physics Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-03-12 03:36:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9099620/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9099620/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105925245,"identity":"8ccb8651-9c65-4f92-95ca-ddf5746c5c1d","added_by":"auto","created_at":"2026-04-01 13:19:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1385397,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of RPL27a increases p53 levels, G1 phase arrest and apoptosis in GC-1 cells. (A-L) RPL27a-siRNA were transfected into GC-1 cells for 48 h. (A) Protein levels, detected by IB. (B) The protein levels in cells of NC was quantitative as 1.0. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3). (C) mRNAs levels were normalized to that of β-actin. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3). (D-F) Detection of RPL27a, p53 and MDM2 after knockdown of RPL27a with IF (magnification, 400×, bar = 10 μm). (G) Histograms of cell cycle percentages. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3).\u0026nbsp;(H) Histograms of apoptosis percentages. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (\u003cem\u003en\u003c/em\u003e = 3). (I)\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e (\u003c/strong\u003eJ) Nuclelolin (red) and B23 (green), (Blue indicates nucleoli, magnification, 400×, bar = 10 μm). (K) and (L) Ribosomal profiles, analyzed by sucrose gradient sedimentation. The absorbance was measured at A254; peaks show 40S, 60S and 80S. NC, negative control; IB, immunoblotting; IF, immunofluorescence; si-RPL27a, RPL27a-siRNA\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/67e2ac5ce1c29945b05655d2.png"},{"id":105925251,"identity":"ce3054d0-d1e9-48a7-b195-dba3711f595e","added_by":"auto","created_at":"2026-04-01 13:19:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1329104,"visible":true,"origin":"","legend":"\u003cp\u003eRPL27a knockdown-induced cell cycle arrest and apoptosis is p53-dependent in GC-1 cells. (A-E) RPL27a and p53-siRNA were co-transfected into GC-1 cells.\u003cstrong\u003e (\u003c/strong\u003eA) The protein level was detected with IB. (B) mRNAs levels were normalized to that of β-actin. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (\u003cem\u003en\u003c/em\u003e = 3). (C-a) The representative images of cell cycle analysis by flow cytometry. (C-b) The representative images of apoptosis by flow cytometry. UR=late apoptosis; LR=early apoptosis. (D) Histograms of cell cycle percentages. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3). (E) Histograms of apoptosis percentages. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3). NC, negative control; IB, immunoblotting; si-RPL27a, RPL27a-siRNA; si-p53, p53-siRNA\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/77db40214fdf5030a944de52.png"},{"id":106093367,"identity":"b62b735c-b7e6-4cec-8a44-2c3584ce31dd","added_by":"auto","created_at":"2026-04-03 11:37:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1503531,"visible":true,"origin":"","legend":"\u003cp\u003eRPL27a knockdown-induced cell cycle arrest and p53 activation is RPL5-dependent in GC-1 cells. (A-E) RPL27a and RPL5-siRNA were co-transfected into GC-1 cells. \u003cstrong\u003e(\u003c/strong\u003eA) The protein level was detected with IB. (B) mRNAs level was normalized to that of β-actin. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3). (C-a) The representative images of cell cycle analysis by flow cytometry. (C-b) The representative images of apoptosis by flow cytometry. UR=late apoptosis; LR=early apoptosis. (D) Histograms of cell cycle percentages. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3). (E) Histograms of apoptosis percentages. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 (\u003cem\u003en\u003c/em\u003e = 3). NC, negative control; IB, immunoblotting; si-RPL27a, RPL27a-siRNA; si-RPL5, RPL5-siRNA\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/3e0e07a0aa3840b580df1fbb.png"},{"id":106093361,"identity":"9b4e8558-e6b3-46aa-afcf-c3e02fecfd1a","added_by":"auto","created_at":"2026-04-03 11:36:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":937447,"visible":true,"origin":"","legend":"\u003cp\u003eProteins interaction and p53 accumulation were verified by Co-IP and in vitro ubiquitination assays. (A) and (B) The analysis of interaction between RPL27a, RPL5 and MDM2 with GST assays. (C) Flag-RPL27a was transfected into GC-1 cells, and IP with anti-Flag, then followed by IB. (D) HA-MDM2 was transfected alone or transfected together with si-RPL27a into GC-1 cells, and IP with anti-HA, then followed by IB. (E) His-Ub plasmids and si-RPL27a were co-transfected into GC-1 cells; IP with anti-His, then followed by IB. IP, Immunoprecipitation; IB, immunoblotting; si-RPL27a, RPL27a-siRNA\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/c62e9a10557248f60ee298bc.png"},{"id":106093374,"identity":"40810051-e06f-43e9-9d66-79beb32c2dc1","added_by":"auto","created_at":"2026-04-03 11:37:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":901996,"visible":true,"origin":"","legend":"\u003cp\u003eThe bindings of RPL27a with RPL5, and MDM2 were verified. (A)\u003cstrong\u003e \u003c/strong\u003eGC-1 cells were transfected with HA-MDM2 (amino acids 1–489) and fragments of Flag-RPL27a, and IP with anti-HA and anti-Flag, then followed by IB. (B) GC-1 cells were transfected with Flag-RPL27a (amino acids 1–148) and fragments of HA-MDM2, and IP with anti-HA and anti-Flag. (C) GC-1 cells were transfected with His-RPL5 (amino acids 1–297) and fragments of Flag-RPL27a, and IP with anti-His and anti-Flag. (D) Flag-RPL27a (amino acids 1–148) and fragments of His-MDM2 was transfected into GC-1 cells, and IP with anti-His and anti-Flag. IP, Immunoprecipitation; IB, immunoblotting\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/3e11634010d64a8fdc81fce2.png"},{"id":106093469,"identity":"cdbfe6f5-fea9-457b-9bf7-d3addb5c0b8d","added_by":"auto","created_at":"2026-04-03 11:37:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3458050,"visible":true,"origin":"","legend":"\u003cp\u003eRPL27a regulates p53 activation in spermatogonia via the RPL5-MDM2-p53 pathway in vivo. (A-G) The testis was separated 3 weeks post injection of adeno-associated virus with shRPL27a. (A) The relative testes weight (testes weight/body weight) (\u003cem\u003en\u003c/em\u003e = 4). (B) The RPL27a mRNA expression detected by Real-time PCR (\u003cem\u003en\u003c/em\u003e = 4). (C) The protein levels were detected by IB. (D) The protein level was quantitative as target protein/β-actin, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (\u003cem\u003en\u003c/em\u003e = 3). (E) The histological observation of H\u0026amp;E staining. Red arrows indicate spermatogonia. (E-a and c), magnification 200 ×, scale bar = 20 μm; (E-b and d), magnification 400 ×, scale bar = 20 μm. (F) Apoptotic detection of spermatogonia by TUNEL. Blank arrows indicate apoptotic spermatogonia. (F-a and c), magnification 200 ×, scale bar = 20 μm; (F-b and d), magnification 400 ×, scale bar = 20 μm. (G) The detection of RPL27a mRNA of spermatogonia by IF, White arrows indicate spermatogonia. (G-a and c), magnification 200 ×, scale bar = 20 μm; (G-b, c, d, f, g and h), magnification 400 ×, scale bar = 20 μm. NC, negative control; KD, Knockdown of RPL27a; IHC, immunohistochemistry; IB, immunoblotting; IF, immunofluorescence; sc, Sertoli cell; sg, spermatogonia; psc, pachytene spermatocyte; lsc, leptotene stage spermatocyte; st, spermaticd; sp, sperm\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/b859e5aad14aedd6cdfa35c3.png"},{"id":106093418,"identity":"beee4841-f85d-40fe-9d69-a85920619324","added_by":"auto","created_at":"2026-04-03 11:37:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3506377,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of proteins were detected by IF in spermatogonia in vivo. (A-D) The testis was separated 3 weeks post injection of adeno-associated virus with shRPL27a, and the expression of proteins were detected by IF; White arrows indicate spermatogonia. (a and e), magnification 200 ×, scale bar = 20 μm; (b, c, d, f, g and h), magnification 400 ×, scale bar = 20 μm\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/86b189f64fdbb6a8cd606bd5.png"},{"id":106401679,"identity":"84937fce-8374-44b0-91a6-a6c030fdf95b","added_by":"auto","created_at":"2026-04-08 09:09:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16669342,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/4ae713a7-3481-4298-81a5-10b4aa5cbe5a.pdf"},{"id":105925246,"identity":"68fd2eef-67da-487e-8714-70d269b236cc","added_by":"auto","created_at":"2026-04-01 13:19:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15527,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/49870bd99ef30294efeb2ff8.docx"},{"id":106093105,"identity":"d9b0aeb6-a06f-4375-be30-d14e0db1f30a","added_by":"auto","created_at":"2026-04-03 11:34:43","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":375602,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.doc","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/8835f12f3998ed18f90cc8a0.doc"},{"id":106093544,"identity":"21661c0c-e751-4269-bc73-b75ee2accb03","added_by":"auto","created_at":"2026-04-03 11:37:56","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22016,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.xls","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/852160841aa37e3f9a460c81.xls"},{"id":106093138,"identity":"6e23708c-b4d7-4c48-a016-b2a33a6785e8","added_by":"auto","created_at":"2026-04-03 11:35:12","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":32714,"visible":true,"origin":"","legend":"","description":"","filename":"ESM3.doc","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/b44ca219e60aa6bf17f82464.doc"},{"id":106093336,"identity":"c04c1edb-cffe-46f5-a20c-c08f15914e14","added_by":"auto","created_at":"2026-04-03 11:36:52","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2851528,"visible":true,"origin":"","legend":"","description":"","filename":"ESM4.doc","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/c176951e212e284eeccc92aa.doc"},{"id":105925254,"identity":"7cee8fae-7060-47b4-99f9-59c855b53f1b","added_by":"auto","created_at":"2026-04-01 13:19:08","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":50688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"ESM5.xls","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/a822a4f214e8072415568d4e.xls"},{"id":105925257,"identity":"62c1e77b-3586-4dac-adaf-8c19dcbb05f6","added_by":"auto","created_at":"2026-04-01 13:19:08","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":134517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9099620/v1/153354face60c6cdb1af4cd3.png"}],"financialInterests":"","formattedTitle":"Loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction in mouse spermatogonia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpermatogonia as an initial status of spermatogenic cells plays an important role in maintaining spermatogenesis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; however, excessive apoptosis of spermatogonia disrupts normal spermatogenesis, thereby resulting in inadequate sperm counts and quality defects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], which is one cause of male infertility [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Infertility involves complex pathogenetic mechanisms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and is affected about 15% of all couples worldwide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, revealing the mechanisms of spermatogonial apoptosis can help understand the process of male infertility.\u003c/p\u003e \u003cp\u003eThe protein p53 is crucial in DNA damage repair, cell cycle arrest and apoptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], is thought to be critical in regulating the apoptosis of spermatogonia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Normal ribosome biogenesis is necessary for maintaining cell homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], but any interference with this complex process can cause ribosomal stress that leads to p53 activation through the interaction between ribosomal proteins (RPs) and MDM2 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A variety of RPs interact with MDM2 and form a RP\u0026ndash;MDM2-p53 multi-complex, which provides a potential link between ribosomal stress and p53 activation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The binding of RPL26 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and RPL23 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] with MDM2 can be enhanced under ribosomal stress, thereby activating p53. RPL5 and RPL11 are effectors of ribosomal stress that, together with MDM2, compose the RPL5/RPL11-MDM2-p53 complex [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Downregulation of RPS19 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], RPL23a [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and RPS27a [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] has been verified to increase p53 activity in an RPL5- or RPL11-dependent manner.\u003c/p\u003e \u003cp\u003eRPL27a is a ribosomal protein constituting the 60S large ribosomal subunit [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies have shown that the deletion of RPL27a causes p53 accumulation and activation through MDM2\u0026ndash;p53 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We have screened RPL27a as a linker between DNA damage and apoptosis of spermatogonia, and we have proposed that loss of RPL27a expression affects p53 accumulation through the RPL5-MDM2-p53 complex [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, RPL27a regulates p53 accumulation whether it is involved in RPL5\u0026ndash;dependent that is still unclear. Thus, the relationship of RPL27a expression and p53 activation in apoptosis of spermatogonia was further investigated in the present study. The interactions of RPL27a and RPL5, of RPL27a and MDM2 were confirmed, which inhibits MDM2-mediated p53 ubiquitination in spermatogonia. Therefore, balanced levels of RPL27a are crucial for maintaining normal levels of p53, and the loss of RPL27a expression enhances the binding of RPL5 and MDM2, thus leading to activation of p53 in spermatogonia. These results provide insight into the relationship of RPs and p53 activation in apoptosis of spermatogonia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal\u003c/h2\u003e \u003cp\u003eLanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou, China) provided the mice for irradiation, and the Obio Biotech (Shanghai, China) provided the mice for AAV experiments. The male C57BL/6 mice were 4 weeks old with body weight 16\u0026ndash;18 g.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTerminal dUTP nick end labeling (TUNEL) assay\u003c/h3\u003e\n\u003cp\u003eApoptotic spermatogenic cells were detected with TUNEL assays (Servicebio, Wuhan, China) according to previous study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the TUNEL reaction was finished, then the sections were stained with 4, 6-diamidino-2-phenylindole (DAPI) (Servicebio) for 10 min at 5 \u0026micro;g/mL and covered with anti-fade mounting medium.\u003c/p\u003e\n\u003ch3\u003eCell culture and transient transfection\u003c/h3\u003e\n\u003cp\u003eGC-1 spermatogonia cells derived from mouse (no. BNCC338398) (BeNa, Culture Collection, Beijing, China) that were cultured in high-glucose DMEM (Meilunbio, Dalian, China) supplemented with 10% fetal bovine serum (ExCell bio, Shanghai, China) under controlled conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). The cells were harvested 48 h after transfection and then treated with lysis buffer for immunoblotting analysis.\u003c/p\u003e\n\u003ch3\u003ePlasmids, drugs, antibodies, and siRNAs\u003c/h3\u003e\n\u003cp\u003eHis-tagged MDM2 and RPL5 expression plasmids were constructed by inserting at \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI sites. The full-length RPL27a was inserted into the pEGX.6P.1 vector, then constructed the GST-RPL27a bacterial expression vector. The RPL27a cDNA was inserted into the pcDNA3.1-3xFlag-C vector, then constructed the overexpression of Flag-tagged RPL27a plasmids. HA-tagged MDM2 and His-tagged ubiquitin B (Ub) expression plasmids were constructed by inserting the full-length MDM2 cDNA and Ub into pUC57 vector, after double digestion and then cloned into the pIRES2-ZsGreen1 vector. The pEX-3-RPL27a cDNA was amplified using the following mRNA primers: 5\u0026prime;-GCTCTAGATTACTTGTCTTCTGGTTTGT-3\u0026prime; and 5\u0026prime;-CCCAAGCTTATGCAGATTTTCGTGAAAAC-3\u0026prime;. The Flag-RPL27a, HA-MDM2, and His-RPL5 fragments were also cloned using PCR and inserted into the pIRES2-ZsGreen1 vector. The plasmid informations were listed in Online Resource 1.\u003c/p\u003e \u003cp\u003eLipo2000 (no. 2359258) was obtained from Invitrogen (Carlsbad, CA, USA), and MG132 (no. HY-13259) was obtained from Medchemexpress (NJ, USA). Antibodies to the following proteins were used for immunoblotting analysis: β-actin (no. ab8227), GAPDH (no. ab181602), RPL27a (no. ab74731), MDM2 (no. ab16895), nucleolin (no. ab129200), p53 (no. ab74731), p21 (no. ab109199), ATM (phospho S1981) (no. ab81292), γH2Ax (phospho S139) (no. ab81299), 53BP1 (no. ab175933), Rad51 (no. ab133534) (Abcam, Cambridge, UK), RPL5 (no. YT4117) and RPL27a (no. YT4103) (Immunoway, NJ, USA), B23 (no. sc-271737 FITC) (Santa Cruz, California, USA) and MDM2 (no. GTX100531) (Genetex, CA, USA), Bax (no. AF0120), Bcl2 (no. AF6139) and Cleaved-Caspase3 (no. AF7022) (Affinity, Melbourne, Australia), and Caspase3 (no. 19677-1-AP) (Proteintech, Wuhan, China). β-actin and GAPDH were used as protein controls to normalize the levels of protein expression. The sequences of RPL27a and p53 were knocked down with siRNAs synthesized by Genepharma (Shanghai, China), as described in previous study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The three different siRNAs to RPL5 were synthesized by Genepharma, as shown in Online Resource 2.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting (IB)\u003c/h3\u003e\n\u003cp\u003eThe protocols of IB was according to our previous study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, a total of 20\u0026ndash;40 \u0026micro;g protein was subjected to electrophoresis and transferred to polyvinylidene difluoride membranes, then blocked with 5% nonfat milk and incubated overnight with primary antibodies at 4\u0026deg;C. Finally, the bands were detected by immunoreactivity..\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle and apoptosis analysis\u003c/h2\u003e \u003cp\u003eThe analysis of cell cycle with propidium iodide (PI) and apoptosis with Annexin V/PI assay by flow cytometer (Beckman Coulter, Inc., CA, USA) according to our previous studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Data were analyzed with the ModFit software program.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of the ribosomal subunit ratio\u003c/h3\u003e\n\u003cp\u003eGC-1 cells were collected as described in a previous study and extracted on ice with 20 mM HEPES (pH 7.4). The samples were cooled in ice water, added to a 5%\u0026ndash;70% sucrose density gradient (20 mM HEPES buffer) and analyzed by the absorbance at 254 nm (Biocomp, CA) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGST-fusion assay\u003c/h3\u003e\n\u003cp\u003eGST-fusion assays were performed according to the protocols [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, the expression plasmids of His-tagged RPL5 and His-tagged MDM2 were transfected into \u003cem\u003eEscherichia coli BL21\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e). Then, the His-RPL5 and His-MDM2 was purified with an Ni\u003csup\u003e2+\u003c/sup\u003e-NTA column (Thermo Fisher Scientific, USA). Finally, the levels of binding proteins were determined by IB with anti-S-Tag and GST antibodies.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (co-IP) and in vitro ubiquitination assay\u003c/h2\u003e \u003cp\u003eGC-1 cells were transfected with tagged plasmids, or vector control after transfection with RPL27a-siRNA for 24 h, the in vitro ubiquitination experiments needed to treat with 40 \u0026micro;M MG132 for 6 h. Subsequently, 70% of the lysate was incubated with anti-Flag, anti-HA or anti-His monoclonal antibody (Cell Signaling Technology, USA), or control IgG [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E, immunofluorescence, immunohistochemistry and in situ hybridization\u003c/h2\u003e \u003cp\u003eThe deparaffinized and dehydrated testicular sections were stained with hematoxylin solution, then were stained with eosin and sealed with neutral gum. The permeabilized testicular sections and GC-1 cells were incubated with primary antibody (1:100), then with secondary antibody: FITC (GB22303; Servicebio)/Cy3 (GB21401; Servicebio) for 1 h at room temperature. The sections and cells were stained for with DAPI (Servicebio) before covering [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The GC-1 cells were scanned and analyzed with a confocal laser microscope (LSM, Carl Zeiss AG, Germany) or observed under a biomicroscope (BX53, Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eThe protocols of immunohistochemistry was described as the previously [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, the incubation of secondary antibody was finished, then the testicular sections were stained with 3,3\u0026acute;-diaminobenzidine. The in situ hybridization assay was according to previous study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The deparaffinized and dehydrated testicular sections were incubated with the digoxigenin-labeled RPL27a cDNA probe sequence 5\u0026prime;- TGCTAATCTGTCCAGTATTAAGTGACCAGTCGTCCT-3\u0026prime; at 42\u0026deg;C for 3 h. Finally, the sections were incubated with IgG secondary antibody labeled Cy3 (GB21401; Servicebio), then stained with DAPI (Servicebio) before covering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR analyses\u003c/h2\u003e \u003cp\u003eThe primers was designed and synthesized by TaKaRa company (Dalian, China), and the DDCt method was used to analyze the level of mRNAs. The primers used were 5\u0026prime;-ATTACCACTTGAAGAGGAACCAGAG-3\u0026prime; and 5\u0026prime;-TTGCCCAGAACTTTGTAGTAGCC-3\u0026prime; (RPL27a); 5\u0026prime;-TGGAGGTGAATGGAGGTGAATAC-3\u0026prime; and 5\u0026prime;- TCAGAGTCATAACCAGGGAATCG-3\u0026prime; (RPL5); 5\u0026prime;-AAGTGTGCCGTTGTCTCTTCG-3\u0026prime; and 5\u0026prime;-AAATCTGTCAGGCTGGTCTGC-3\u0026prime; (p21); 5\u0026prime;-CCATCTACAAGAAGTCACAGCAC-3\u0026prime; and 5\u0026prime;-GCCATCACCATCGGAGCAG-3\u0026prime; (p53); 5\u0026prime;-GGTTGAGGGCCACCTTATTT-3\u0026prime; and 5\u0026prime;-GAAGAAAGACCGGGAAGAGAAA-3\u0026prime; (47S pre-rRNA); 5\u0026prime;-GGCAGGGGAGAGTGATACAGA-3\u0026prime; and 5\u0026prime;-AAGTCAAAGTTCCACCGTTCTCG-3\u0026prime; (MDM2) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRPL27a knockdown in vivo\u003c/h2\u003e \u003cp\u003eAn adeno-associated virus (AAV) was used to knock down RPL27a in vivo. The mouse RPL27a cDNA (NM_011975.3) was cloned into AAV expression plasmids with the primers described in Online Resource 3. The recombinant AAV serotype-PHP.eB (AAV-PHP.eB) was produced by transfection of AAV expression plasmids into human embryonic kidney 293 cells. Male mice were used for experiments and subjected to injections as previously reported [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The mice were anaesthetized with isoflurane, and administered 5\u0026times;10\u003csup\u003e11\u003c/sup\u003e vgs virus in a 300 \u0026micro;l volume by tail-vein injection. The animals were sacrificed 3 weeks post injection, and testes were dissected for analysis. The viruses infecting the seminiferous tubules of the testes were observed and imaged to detect GFP expression in frozen sections (Online Resource 4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll testicular sections were scanned and analyzed in Panoramic MIDI software (3DHISTECH, Budapest, Hungary). GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The statistical differences between two groups were analyzed by student\u0026rsquo;s \u003cem\u003et-\u003c/em\u003etest. Multiple groups of statistical analysis were assessed with one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s test. Data are indicated as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 were considered thresholds of statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of RPL27a induces p53 activation, promotes apoptosis and cell cycle arrest in GC-1 cells\u003c/h2\u003e \u003cp\u003eWe analyzed the effect of RPL27a knockdown on apoptosis, cell cycle arrest, and expression of RPL5, p53 and MDM2 in GC-1 cells. The levels of RPL5 mRNA by different siRNAs is shown in Online Resource 5. The expression of p53 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B) and mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), as well as the p53 target genes \u003cem\u003ep21\u003c/em\u003e and \u003cem\u003eMDM2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), increased significantly by knockdown of RPL27a. The fluorescence signals of RPL27a protein were weakened in the nucleoli and cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), whereas the fluorescence signals of p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and MDM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) were enhanced in the nucleus and cytoplasm. These results indicated that the decreased level of RPL27a resulted in activation of p53 and p53 target genes \u003cem\u003ep21\u003c/em\u003e and \u003cem\u003eMDM2\u003c/em\u003e, and this process was associated with RPL5. The activation of p53 induced apoptosis, whereas the knockdown of RPL27a significantly increased the apoptotic ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Consistently with these findings, RPL27a knockdown significantly increased the ratio of cells in G1 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), thereby indicating that RPL27a knockdown activates p53 and causes cell cycle arrest, which leads to spermtogonial apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of RPL27a alters the ribosomal subunit ratio of GC-1 cells\u003c/h2\u003e \u003cp\u003eRPL27a knockdown decreased the expression of 47S pre-rRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), thereby indicating that knockdown of RPL27a inhibited rRNA synthesis. Nucleolin (NCL) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and nucleophosmin (B23) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], two nucleolar integrity marker proteins, were selected to determine the effect of RPL27a knockdown on nucleolar integrity. RPL27a knockdown increased the fluorescence signal of NCL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) and B23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ); thus, the nucleolar structure in GC-1 cells may be damaged by RPL27a knockdown. The 80S:60S and 80S:40S ratio decreased in the RPL27a knockdown cells that analyzed by polysome profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Therefore, RPL27a knockdown changes the ribosomal subunit ratio and could generate nucleolar stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRPL27a knockdown-caused cell cycle arrest and apoptosis is p53-dependent, which requires RPL5\u003c/h2\u003e \u003cp\u003eGC-1 cells were co-transfected with RPL27a-siRNA and p53-siRNA to determine whether the G1 phase arrest induced by RPL27a knockdown was p53 dependent. The RPL27a knockdown-induced increases in p21 and MDM2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the p21 mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which eliminated by co-transfected with p53-siRNA. The representative images of flow cytometry in GC-1 cells by co-transfected with si-p53 and RPL27a were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. RPL27a knockdown-induced G1 phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), which eliminated by co-transfected with p53-siRNA. Therefore, RPL27a knockdown-caused cell cycle arrest and apoptosis is p53-dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGC-1 cells were co-transfected with RPL27a-siRNA and RPL5-siRNA to determine whether the p53 activation caused by RPL27a knockdown was RPL5 dependent. The RPL27a knockdown-induced increases in p21 and MDM2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and the p21 mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which eliminated by co-transfected with RPL5-siRNA. The representative images of flow cytometry in GC-1 cells by co-transfected with si-RPL5 and RPL27a were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. RPL27a knockdown-induced G1 phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), which eliminated by co-transfected with RPL5-siRNA. Therefore, RPL27a knockdown activates p53 activation in RPL5-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of RPL27a enhances the RPL5 and MDM2 interaction\u003c/h2\u003e \u003cp\u003eGST-fusion assays demonstrated that RPL27a co-interacted with MDM2 and RPL5 \u003cem\u003ein vitro\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB show that purified His-RPL5 and His-MDM2 were bound by purified GST-RPL27a protein but not GST alone. These results demonstrated that RPL27a binds both with RPL5 and MDM2 in GC-1 cells.\u003c/p\u003e \u003cp\u003eThe interaction of RPL27a with MDM2 and of RPL27a with RPL5 ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC); lane 6 compared to lane 3 in lower panel) was enhanced. The above results suggested that RPL27a binds RPL5 and MDM2, and exogenous overexpression of RPL27a promotes their interaction. In addition, GC-1 cells were transfected together with HA-MDM2 and RPL27a-siRNA, and the protein interaction and expression were analyzed with co-IP and IB. The interaction of MDM2 with RPL5 ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD); lane 9 compared to lane 8 in middle panel) was enhanced, but the interaction of MDM2 with RPL27a ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD); lane 9 compared to lane 8 in lower panels) was weakened by RPL27a knockdown. These results suggested that MDM2 interacts with RPL27a and RPL5; when RPL27a was knocked down, the interaction between RPL5 and MDM2 was promoted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of RPL27a stabilizes p53\u003c/h2\u003e \u003cp\u003eHis-Ub plasmids and RPL27a-siRNA was transfected into GC-1 cells to analyze the effect of RPL27a knockdown on p53 ubiquitination. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE shows that RPL27a knockdown partially rescued the p53 accumulation (lane 3 compared to lane 2 in upper panel), and the expression of RPL5, p53 and MDM2 increased after RPL27a knockdown ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE); lane 3 compared to lane 2). These results suggested that RPL27a knockdown inhibits MDM2-mediated ubiquitination of p53.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eRPL27a interacts with RPL5 and MDM2, according to the Co-IP assay in vitro\u003c/h2\u003e \u003cp\u003eHA-MDM2, Flag-RPL27a and His-RPL5 plasmids were constructed and cloned with different fragments to determine the sites responsible for the binding between RPL27a and MDM2, and co-IP assays were performed. Full-length MDM2 (amino acids 1\u0026ndash;489, molecular weight is about 55 KDa) bound to full-length RPL27a (amino acids 1\u0026ndash;148) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and full-length RPL27a (amino acids 1\u0026ndash;148) bound to full-length MDM2 (amino acids 1\u0026ndash;489) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Full-length RPL5 (amino acids 1\u0026ndash;297) bound to the full-length RPL27a (amino acids 1\u0026ndash;148) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and full-length RPL27a (amino acids 1\u0026ndash;148) bound to full-length RPL5 (amino acids 1\u0026ndash;297) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Together, these results further suggested that RPL27a interacts both with RPL5 and MDM2.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eRPL27a interacts both with RPL5 and MDM2, on the basis of molecular docking\u003c/h2\u003e \u003cp\u003eThe hydrogen bonding of the binding interface of RPL5-RPL27a and MDM2-RPL27a was analyzed as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF. The specific amino acid information for hydrogen bonding and hydrophobic interaction is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF. The analysis of the interaction of amino acids at the binding interface between MDM2 and RPL27a revealed that hydrogen bonding and electrostatic interaction were important interactions. The results of protein-protein docking further suggested interactions between RPL27a and RPL5, and RPL27a and MDM2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of RPL27a promotes p53 accumulation in spermatogonia in vivo\u003c/h2\u003e \u003cp\u003eAAV was used to knock down RPL27a in vivo for 3 weeks, then the apoptotic spermatogonia, RPL27a, p53, MDM2 and RPL5 levels in spermatogonia in vivo were analyzed. The decrease in relative testis weight was showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and the significant decrease in RPL27a mRNA expression detected by Real-time PCR in the shRPL27a testes in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. Treatment of AAV with shRPL27a increased the RPL5, p53 and MDM2 levels in testes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Histological observations showed vacuolization of the seminiferous tubules, disordered spermatogenic cells, and diminished spermatogenic cell numbers and types in shRPL27a groups compared with control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The apoptotic cells were clearly more abundant in the shRPL27a groups than the control groups Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Moreover, the RPL27a mRNA expression detected by in situ hybridization in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG.\u003c/p\u003e \u003cp\u003eThe weak fluorescence signal of RPL27a was observed in spermatogonia with knockdown of RPL27a of testis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In addition, relatively strong staining of p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), MDM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and RPL5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) was observed in spermatogonia with knockdown of RPL27a of testis. These results indicated that RPL27a knockdown induced apoptosis of spermatogonia via the accumulation of p53 through RPL5-MDM2-p53 pathway in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, RPL27a knockdown induced an increase in NCL and B23, thereby indicating generation of ribosomal stress consistent with IR-induced movement of NCL into the nucleoplasm, and decreased RPL27a expression in the nucleoli and cytoplasm [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, real-time PCR indicated that knockdown of RPL27a decreased the expression of 47S pre-rRNA, thereby inhibiting rRNA synthesis. Therefore, RPL27a knockdown might trigger ribosomal stress, thereby leading to p53 activation.\u003c/p\u003e \u003cp\u003eA variety of RPs bind MDM2 and form RP-MDM2-p53 complexes, thus, providing a potential link between ribosomal stress and activation of p53 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Their decreased expression levels increase p53 activity in a manner dependent on RPL5 or RPL11 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Because RPL5 and RPL11 are nucleolar stress effectors and sensors, the RPL5/RPL11-MDM2-p53 complex is the classical model of RP and p53 binding [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, co-knockdown of p53 eliminated cell cycle arrest and apoptosis caused by RPL27a knockdown, thus, indicating that the cell cycle arrest and apoptosis caused by RPL27a knockdown was p53 dependent. Moreover, the knockdown of RPL27a caused p53 accumulation in a manner that was RPL5 dependent, because co-knockdown with RPL5 eliminated p53 activation, and attenuated the cell cycle arrest and apoptosis induced by RPL27a knockdown.\u003c/p\u003e \u003cp\u003eIn this study, RPL27a binds both to MDM2 and RPL5 was verified in spermatogonia by GST assay. In addition, in vitro ubiquitination showed that the knockdown of RPL27a significantly inhibited MDM2-mediated p53 ubiquitination and led to p53 activation; consequently, RPL27a is crucial in negatively regulating p53 activation in spermatogonia. Furthermore, Co-IP experiments were used to further confirm the binding domain that RPL27a bound RPL5 and MDM2, although which amino acid sequences bound each other was unclear, possibly because the linear binding experiment could not fully simulate the true spatial interaction between proteins. However, the protein-protein docking results indicated that both RPL5 and MDM2 formed a stable complex structure with RPL27a. Finally, we confirmed that the knockdown of RPL27a promoted the binding of MDM2 and RPL5, and inhibited MDM2-mediated p53 ubiquitination. The RPL27a knockdown by AAV assay further clarified the promotion of spermatogonia apoptosis via the accumulation of p53 in vivo.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides further confirmation that RPL27a is a novel regulator of the RPL5-MDM2-p53 complex, and indicate that the loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction and inducing cell cycle arrest and apoptosis of spermatogonia. Our results not only confirmed the binding between RPL27a and the RPL5-MDM2 interaction, but also revealed RPL27a as a novel regulator of the RPL5-MDM2-p53 pathway. Our findings provide a better understanding of the relationship of RPs and p53 activation in the apoptosis of spermatogonia.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e4, 6-diamidino-2-phenylindole (DAPI); Ribosomal proteins (RPs); Phosphate buffered saline (PBS); polyvinylidene difluoride membrane (PVDF); Real-time quantitative polymerase chain reaction (PCR).\u0026nbsp;\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially funded by the Youth Talent Program of \u0026ldquo;Fuxi\u0026rdquo; (Gaufx-03Y02), the National Natural Science Foundation of China (11875061, 31902339), the Scientific Research Start-up Funds for Openly-Recruited Doctors of Gansu Agricultural University (2017RCZX-13), the Talent Project of Longyuan (Gansu province), the fund of College of Veterinary Medicine of Gansu Agricultural University (2025DY-01), Youth Innovation and Entrepreneurship (E339221SR0), the Foundation for Youth Doctor of Gansu Province (2021QB-026), the Special Funds for Discipline Construction of Gansu Agricultural University (GAU-XKJS-2018-067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially funded by the National Natural Science Foundation of China (11875061, 12005042,\u0026nbsp;31902339), the Youth Talent Program of \u0026ldquo;Fuxi\u0026rdquo; (Gaufx-03Y02),\u0026nbsp;the Scientific Research Start-up Funds for Openly-Recruited Doctors of Gansu Agricultural University (2017RCZX-13),\u0026nbsp;the Talent Project of Longyuan (Gansu province) Youth Innovation and Entrepreneurship (E339221SR0),\u0026nbsp;the Foundation for Youth Doctor of Gansu Province (2021QB-026),\u0026nbsp;the Special Funds for Discipline Construction of Gansu Agricultural University (GAU-XKJS-2018-067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare. All authors read and approved the\u0026nbsp;\u003c/p\u003e\n\u003cp\u003efinal manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe management and euthanasia of laboratory animals complied with the Administration of Affairs Concerning Experimental Animals of the Ministry of Science and Technology of China (1988.11.14), and carried out in accordance with UK Home Office regulations (UK Animals Scientific Procedures Act of 1986; project license PPL PD8CBD97C). Animal experiments were approved by the Ethics Committee and Institutional Animal Care and Use Committee (IACUC) of the Institute of Modern Physics, Chinese Academy of Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAvailability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll related data not included in the manuscript will be available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYuxuan He\u003c/strong\u003e: Investigation, Methodology, Formal analysis, Validation, Visualization, Software, Data curation, Writing\u0026mdash;original draft, Writing\u0026mdash;review and editing. \u003cstrong\u003eRong Gan\u003c/strong\u003e: Investigation, Software, Validation, Visualization, Writing\u0026mdash;original draft. \u003cstrong\u003eJiahao Zhang\u003c/strong\u003e: Investigation, Methodology, Validation, Visualization. \u003cstrong\u003eWeitao Dong\u003c/strong\u003e: Software, Validation, Visualization. \u003cstrong\u003eDong Zhang and Xiaoyun Pang\u003c/strong\u003e: Investigation, Methodology, Resources. \u003cstrong\u003eYanyan Wang and Li Xue\u003c/strong\u003e 7: Investigation, Validation. \u003cstrong\u003eJincheng Yang and Rui Luo\u003c/strong\u003e: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Validation, Visualization, Writing\u0026mdash;original draft, Writing\u0026mdash;review and editing. \u003cstrong\u003eRong Li\u003c/strong\u003e: Resources, Validation, Supervision. \u003cstrong\u003eXianghong Du\u003c/strong\u003e: Conceptualization, Funding acquisition, Supervision, Project administration, Writing\u0026mdash;review and editing. \u003cstrong\u003eHongyan Li\u003c/strong\u003e: Conceptualization, Methodology, Supervision, Project administration, Writing\u0026mdash;review and editing.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCao J, Lin ZB, Tong MH, Zhang YL, Li YP et al (2022) Mechanistic target of rapamycin kinase (Mtor) is required for spermatogonial proliferation and differentiation in mice. Asian J Androl 22:169\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/aja.aja_14_19\u003c/span\u003e\u003cspan address=\"10.4103/aja.aja_14_19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBejarano I, Rodr\u0026iacute;guez AB, Pariente JA (2018) Apoptosis is a demanding selective tool during the development of fetal male germ cells. Front Cell Dev Biol 6:65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fcell.2018.00065\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2018.00065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarjault HB, Allemand I (2016) Consequences of irradiation on adult spermatogenesis: Between infertility and hereditary risk. Mutat Res Rev Mutat Res 770(Pt B 340\u0026ndash;348. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mrrev.2016.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.mrrev.2016.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrefumo F, Rossi AC (2018) Endometriosis, endometrioma, and ART results: Current understanding and recommended practices. Best Pract Res Clin Obstet Gynaecol 51:34\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bpobgyn.2018.01.09\u003c/span\u003e\u003cspan address=\"10.1016/j.bpobgyn.2018.01.09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang C, Yao C, Ji Z, Zhao L, Chen H, Li P et al (2021) RNA-binding protein ELAVL2 plays post-transcriptional roles in the regulation of spermatogonia proliferation and apoptosis. Cell Prolif 54:e13098. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/cpr.13098\u003c/span\u003e\u003cspan address=\"10.1111/cpr.13098\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHafner A, Bulyk ML, Jambhekar A, Lahav G (2019) The multiple mechanisms that regulate p53 activity and cell fate. Nat Rev Mol Cell Biol 20:199\u0026ndash;210. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41580-019-0110-x\u003c/span\u003e\u003cspan address=\"10.1038/s41580-019-0110-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Morais MP, Curado RF, Silva KSE, Moura KK, Arruda JT (2016) Male idiopathic infertility and the TP53 polymorphism in codon 72. Genet Mol Res 15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4238/gmr15048882\u003c/span\u003e\u003cspan address=\"10.4238/gmr15048882\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao PY, Yao RQ, Zhang ZC, Zhu SY, Li YX, Ren C et al (2022) Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases. Int J Biol Sci 18:2497\u0026ndash;2514. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.7150/ijbs.70955\u003c/span\u003e\u003cspan address=\"https://doi:10.7150/ijbs.70955\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang K, Wang M, Zhao Y, Sun X, Yang Y, Li X et al (2016) A redox mechanism underlying nucleolar stress sensing by nucleophosmin. Nat Commun 7:13599. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms13599\u003c/span\u003e\u003cspan address=\"10.1038/ncomms13599\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Deisenroth C, Zhang Y (2016) RP-MDM2-p53 pathway: Linking ribosomal biogenesis and tumor surveillance. Trends Cancer 2:191\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.trecan.2016.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.trecan.2016.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang J, Yuan Y, Zhang W, Guan W, Wu Z et al (2010) Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26. Nucleic Acids Res 38:6544\u0026ndash;6554. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkq536\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkq536\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe S, Fujiyama H, Takafuji T, Kayama K, Matsumoto M, Nakayama KI et al (2018) GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system. J Cell Sci 131:jcs213009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jcs.213009\u003c/span\u003e\u003cspan address=\"10.1242/jcs.213009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBursać S, Brdovčak MC, Pfannkuchen M, Orsolić I, Golomb L, Zhu Y et al (2012) Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress. Proc Natl Acad Sci U S A 109:20467\u0026ndash;20472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1218535109\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1218535109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia Q, Zhang Q, Zhang Z, Wang Y, Zhang W, Zhou Y et al (2013) Transcriptome analysis of the zebrafish model of Diamond-Blackfan anemia from RPS19 deficiency via p53-dependent and -independent pathways. PLoS ONE 8:e71782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0071782\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0071782\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe YX, Guo YX, Zhang Y, Hu JJ, Dong WT, Du XH et al (2021) Ionizing radiation-induced RPL23a reduction regulates apoptosis via RPL11-MDM2-p53 pathway in mouse spermatogonia. Biomed Environ Sci 34:789\u0026ndash;802. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3967/bes2021.108\u003c/span\u003e\u003cspan address=\"10.3967/bes2021.108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Zhang H, Huang G, Bing Z, Xu D, Liu J et al (2022) Loss of RPS27a expression regulates the cell cycle, apoptosis, and proliferation via the RPL11-MDM2-p53 pathway in lung adenocarcinoma cells. J Exp Clin Cancer Res 41:33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13046-021-02230-z\u003c/span\u003e\u003cspan address=\"10.1186/s13046-021-02230-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerzian T, Dumble M, Arbab F, Thaller C, Donehower LA, Lozano G et al (2011) Rpl27a mutation in the sooty foot ataxia mouse phenocopies high p53 mouse models. J Pathol 224:540\u0026ndash;552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/path.2891\u003c/span\u003e\u003cspan address=\"10.1002/path.2891\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlkhatabi HA, McLornan DP, Kulasekararaj AG, Malik F, Seidl T, Darling D et al (2016) RPL27A is a target of miR-595 and may contribute to the myelodysplastic phenotype through ribosomal dysgenesis. Oncotarget 7:47875\u0026ndash;47890. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/oncotarget.10293\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.10293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Zhang H, Huang G, Dou Z, Xie Y, Si J et al (2020) Heavy ion radiation-induced DNA damage mediates apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway in mouse spermatogonia. Ecotoxicol Environ Saf 201:110831. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoenv.2020.110831\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2020.110831\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, He Y, Yan J, Zhao Q, Di C, Zhang H (2016) Comparative proteomics reveals the underlying toxicological mechanism of low sperm motility induced by iron ion radiation in mice. Reprod Toxicol 65:148\u0026ndash;158. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.reprotox.2016.07.014\u003c/span\u003e\u003cspan address=\"10.1016/j.reprotox.2016.07.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong CC, Traynor D, Basse N, Kay RR, Warren AJ (201) Defective ribosome assembly in Shwachman-Diamond syndrome. Blood 118:4305\u0026ndash;43122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2011-06-353938\u003c/span\u003e\u003cspan address=\"10.1182/blood-2011-06-353938\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X, Hao Q, Liao J, Zhang Q, Lu H (2013) Ribosomal protein S14 unties the MDM2-p53 loop upon ribosomal stress. Oncogene 32:388\u0026ndash;396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/onc.2012.63\u003c/span\u003e\u003cspan address=\"10.1038/onc.2012.63\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Q, Zhang S, Sun Z, Guo X, Zhou H (2019) E3 ubiquitin ligase Nedd4 is a key negative regulator for non-canonical inflammasome activation. Cell Death Differ 26:2386\u0026ndash;2399. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41418-019-0308-7\u003c/span\u003e\u003cspan address=\"10.1038/s41418-019-0308-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Wei J, Xu K, Sun X, Zhang H, Xiong C (2019) CSE1L participates in regulating cell mitosis in human seminoma. Cell Prolif 52:e12549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/cpr.12549\u003c/span\u003e\u003cspan address=\"10.1111/cpr.12549\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Yu M, Liu C, Zhu H, He X, Peng S et al (2013) miR-34c works downstream of p53 leading to dairy goat male germline stem-cell (mGSCs) apoptosis. Cell Prolif 46:223\u0026ndash;231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/cpr.12013\u003c/span\u003e\u003cspan address=\"10.1111/cpr.12013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui D, Li L, Lou H, Sun H, Ngai SM, Shao G et al (2014) The ribosomal protein S26 regulates p53 activity in response to DNA damage. Oncogene 33:2225\u0026ndash;2235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/onc.2013.170\u003c/span\u003e\u003cspan address=\"10.1038/onc.2013.170\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajasekaran S, Thatte J, Periasamy J, Javali A, Jayaram M, Sen D et al (2018) Infectivity of adeno-associated virus serotypes in mouse testis. BMC Biotechnol 18:70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12896-018-0479-1\u003c/span\u003e\u003cspan address=\"10.1186/s12896-018-0479-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWestdorp KN, Sand A, Moorman NJ, Terhune SS (2017) Cytomegalovirus late protein pUL31 alters pre-rRNA expression and nuclear organization during infection. J Virol 91:e00593\u0026ndash;e00517. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/JVI.00593-17\u003c/span\u003e\u003cspan address=\"10.1128/JVI.00593-17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvitabile D, Bailey B, Cottage CT, Sundararaman B, Joyo A, McGregor M et al (2011) Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin. Proc Natl Acad Sci U S A 108:6145\u0026ndash;6150. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1017935108\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1017935108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Poyurovsky MV, Li Y, Stahl J, Jacq X, Prives C (2009) Ribosomal protein S7 is both a regulator and a substrate of MDM2. Mol Cell 35:316\u0026ndash;326. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molcel.2009.07.014\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2009.07.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong X, Zhao Y, He H, Sun Y (2011) Ribosomal protein S27-like and S27 interplay with p53-MDM2 axis as a target, a substrate and a regulator. Oncogene 30:1798\u0026ndash;1811. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/onc.2010.569\u003c/span\u003e\u003cspan address=\"10.1038/onc.2010.569\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolmberg Olausson K, Nist\u0026eacute;r M, Lindstr\u0026ouml;m MS (2012) p53-dependent and -independent nucleolar stress responses. Cells 1:774\u0026ndash;798. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells1040774\u003c/span\u003e\u003cspan address=\"10.3390/cells1040774\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Apoptosis, Protein interaction, Ribosomal stress, Ribosomal protein L27a","lastPublishedDoi":"10.21203/rs.3.rs-9099620/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9099620/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRibosomal proteins (RPs) regulate p53 activation, which is mediated by mouse double minute 2 homolog (MDM2), ribosomal protein L5 (RPL5) and/or ribosomal protein L11. Thus, this study aimed to explore whether ribosomal protein L27a (RPL27a) interacts with RPL5 and regulates p53 activation in mouse spermatogonia. Immunohistochemistry and immunofluorescence were used to analyze protein expression\u003cem\u003e in vivo\u003c/em\u003e. Protein and mRNA expression was analyzed by immunoblotting and real-time polymerase chain reaction, respectively. The bindings of RPL27a with RPL5, and MDM2 were verified by molecular docking and glutathione S-transferase (GST)-fusion protein assays. Co-immunoprecipitation and in vitro ubiquitination assays were used to confirm protein interactions and p53 accumulation. An adeno-associated virus was used to knock down RPL27a \u003cem\u003ein vivo\u003c/em\u003e. The results showed that knockdown of RPL27a weakened the binding of RPL27a to RPL5 and MDM2, but promoted the interaction between RPL5 and MDM2. Meanwhile, knockdown of RPL27a induced p53-dependent cell cycle arrest and RPL5-dependent p53 activation in mouse spermatogonial GC-1 spg cells. Moreover, RPL27a knockdown induced apoptosis of spermatogonia via the accumulation of p53, as verified \u003cem\u003ein vivo\u003c/em\u003e. This study indicates that RPL27a negatively regulates p53 activation via enhancing the RPL5-MDM2 interaction in spermatogonia, and provides a better understanding of male infertility induced apoptosis of spermatogonia.\u003c/p\u003e","manuscriptTitle":"Loss of RPL27a expression promotes p53 activation by modulating the RPL5-MDM2 interaction in mouse spermatogonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 13:18:56","doi":"10.21203/rs.3.rs-9099620/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-26T18:17:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T14:27:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T13:08:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2026-03-16T10:21:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5779d31-b07b-4ef3-b90f-09aa0af0d1c9","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T13:18:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 13:18:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9099620","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9099620","identity":"rs-9099620","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}