Mouse strains

C57BL/6 N and ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The generation of Bcas2Fl/Fl mice has been described previously [25]. Stra8-GFPCre mice were kindly provided by Prof. Minghan Tong (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) [38, 39]. To generate Bcas2 cKO mice, Stra8-GFPCre mice were crossed with Bcas2Fl/Fl mice. The primers used for PCR to genotype Bcas2Fl/Fl and Stra8-GFPCre mice are shown in Table S4. All mice were bred and housed under specific pathogen-free conditions with controlled temperature (22 ± 1 °C) and exposed to a constant 12-hour light-dark cycle in the animal facilities of China Agricultural University. All experiments were conducted according to the guidelines and with the approval of the Institutional Animal Care and Use Committee of China Agricultural University (No. AW80401202-3-2).

Fertility test

For a duration of 15 days, two 8-week-old ICR female mice were housed together with an 8-week-old male mouse, either Ctrl or cKO. Female mice were examined for the presence of vaginal plugs, and those with plugs were individually housed, with meticulous recording of the dates. The male mice were maintained in separate cages after two days of cohabitation. Daily records were maintained for the number of pups born to each female, along with the respective dates of parturition.

Immunostaining and histologic analysis

The mouse testes were fixed using 4% paraformaldehyde (P6148-500G, Sigma–Aldrich, Missouri, USA) in PBS (pH 7.4) at 4 °C overnight. Subsequently, the samples underwent dehydration through graded ethanol solutions, vitrification with xylene, and embedding in paraffin. Testis sections, cut at a thickness of 5 μm, were utilized for immunostaining and histologic analysis. For histological assessment, sections were dewaxed in xylene, rehydrated in graded ethanol solutions, and stained with H&E. Following sealing with neutral resin, imaging was performed using a Ventana DP200 system.

For immunofluorescence analysis, antigen retrieval was achieved by microwaving the sections with sodium citrate buffer (pH 6.0). Following blocking with 10% goat serum at room temperature for 1 h, the sections were incubated with primary antibodies in 5% goat serum (Table S5) overnight at 4 °C. After PBS washing, the sections were exposed to secondary antibodies (Table S5) at room temperature in the dark for 1 h. The slides were mounted using an antifade mounting medium with DAPI (P0131, Beyotime, Shanghai, China). Imaging was conducted using a Nikon A1 laser scanning confocal microscope and a Zeiss OPTOME fluorescence microscope.

TUNEL apoptosis analysis

Adult testes sections were processed according to the TUNEL Apoptosis Assay Kit protocol (C1088, Beyotime, Shanghai, China). Photomicrographs were acquired using a Nikon A1 laser scanning confocal microscope and a Zeiss OPTOME fluorescence microscope. The quantification of TUNEL+ tubules was performed to assess the percentage of apoptotic cells in both cKO and Ctrl adult mouse testes.

Surface spreading

Testis surface spreading was carried out following established procedures [24, 46]. In brief, half of the mouse testes were isolated and treated with 300 µl of TrypLE (12604021, Thermo, New York, US) at 37 °C for 10 min under constant agitation. The digestion process was halted by adding 30 µl fetal bovine serum (C0235, Beyotime, Shanghai, China). After centrifugation at 1000 rpm for 1 min, the supernatant was discarded. The cells underwent treatment with 300 µl hypotonic buffer (30 mM Tris-HCl, 17 mM trisodium citrate dihydrate, 50 mM sucrose, 5 mM EDTA, 0.5 mM DTT, pH 8.2) containing protease inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China) for 30 min. Slides were pretreated with 20 µl of fixation buffer (1% paraformaldehyde, pH 9.2 with 50 mM boric acid) containing 0.15% Triton X-100 (T9284, Sigma, Missouri, USA) applied evenly on slides in advance. Subsequently, 20 µl aliquots of the cell suspension were dripped onto the slides, which were then incubated at 37 °C for 4 h or overnight in a humidified box. The samples were left to dry at room temperature, and immunofluorescence staining was performed according to the protocol described above.

Flow cytometry

Following established protocols, STRA8+ cell suspensions were isolated and prepared from mouse testes [24]. Initially, adult Ctrl and cKO testes were subjected to digestion with 500 µl of collagenase IV (1 mg/mL, 40510ES60, Yeasen, Shanghai, China) and DNase I (1 mg/mL, 10104159001, Roche, Mannheim, Germany) at 37 °C for 10 min under constant agitation. Subsequently, the testes were further digested for an additional 10 min after replacing collagenase IV with TrypLE. Finally, STRA8+ cell suspensions from Ctrl and cKO mice were sorted using a FACSAria Fusion (BD Biosciences).

RT–PCR and RT–qPCR

Total RNA extraction was performed using RNAiso Plus (9109, Takara, Kusatsu, Japan) and a Direct-zol RNA MicroPrep kit (R2060, Zymo Research, California, USA), with concentration measured using a Nano-300 ultramicro spectrophotometer (Allsheng, Hangzhou, China). Subsequently, cDNA was synthesized following the instructions of the TIANScript II RT kit (KR107, TIANGEN, Beijing, China). The expression levels of target gene transcripts were quantified using a Light Cycle® 96 instrument (Roche) with Hieff UNICON SYBR green master mix (11198ES08, Yeasen, Shanghai, China). AS analyses were conducted on a RePure-A PCR instrument (BIO-GENER, Hangzhou, China). Primers, synthesized by Sangon Biotech, are listed in Table S4. Gapdh or Actb expression served as the control, with its value set as 1. Relative transcript expression levels for other samples were determined by comparing them with the control results.

RNA-seq

Total RNA extraction from STRA8+ cells followed the outlined protocol. In brief, mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads. Following fragmentation, a transcriptome sequencing library was constructed, and library quality was assessed on an Agilent Bioanalyzer 2100 system. Index-coded sample clustering was conducted on a cBot Cluster Generation System using a TruSeq PE Cluster kit v3-cBot-HS (Illumina), following the provided instructions. Subsequently, the library preparations were sequenced on the Illumina NovaSeq platform, generating 150 bp paired-end reads. After quality control, all downstream analyses were performed on clean, high-quality data.

The reference genome index was established, and HISAT2 software (version 2.0.5) aligned paired-end clean reads to the reference genome. FeatureCounts (version 1.5.0) was employed to count the reads mapped to each gene. Subsequently, the fragments per kilobase million (FPKM) value of each gene was calculated based on the gene length and the read count mapped to it. The DESeq2 R package (version 1.20.0) conducted differential expression analysis of cKO/Ctrl STRA8+ cells (three biological replicates per condition). Genes with a padj < 0.05 and |log2FoldChange| > 1, as identified by DESeq2, were considered differentially expressed.

AS analysis

The rMATS software (version 3.2.5) was employed for the analysis of AS events in cKO mouse germ cells utilizing RNA-seq data. The software identified five types of AS events, including SE, RI, MXE, A5SS, and A3SS. The threshold set for screening differentially significant AS events was a false discovery rate (FDR) of less than 0.05. Splicing events with an FDR less than 0.05 and an inclusion-level difference showing significance of at least 5% (0.05) were considered statistically significant. To confirm and visualize AS events based on RNA-seq data, the IGV (Version 2.16.0) was utilized.

GO term enrichment analysis

The GO enrichment analysis for both differentially expressed genes and AS genes was conducted using the clusterProfiler R package (version 3.4.4). This analysis corrected for gene length bias. The mouse genome data (GRCm38/mm10) served as the reference, and the Benjamini–Hochberg method was applied for multiple testing correction. This approach helps identify and interpret the biological processes, molecular functions, and cellular components associated with the genes of interest.

Western blotting

Total protein extraction was performed using cell lysis buffer (P0013, Beyotime, Shanghai, China), supplemented with PMSF (1:100, ST506, Beyotime, Shanghai, China) and a protease inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China). The protein concentration was measured using a BCA protein assay kit (CW0014S, CWBiotech, Beijing, China). Subsequently, protein lysates were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, Ireland). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies (Table S5) overnight at 4 °C. Following this, the membranes were exposed to secondary antibodies (Table S5) at room temperature for 1 h. Protein visualization was achieved using the Tanon 5200 chemiluminescence imaging system following incubation with BeyoECL Plus (P0018S, Beyotime, Shanghai, China).

IP, IP-MS, and Co-IP

As previously outlined, total protein was extracted using cell lysis buffer (P0013, Beyotime, Shanghai, China) supplemented with PMSF (1:100, ST506, Beyotime, Shanghai, China) and a protease inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China) [23, 24]. After 20 min of incubation on ice, the lysate was combined with 20 µl of protein A agarose (P2051-2 ml, Beyotime, Shanghai, China) for pre-clearing at 4 °C for 1 h. Subsequently, 2 micrograms of a BCAS2 antibody (PA2466, Abmart, Shanghai, China) and a normal rabbit IgG (A7016, Beyotime, Shanghai, China) were added to the lysate, followed by overnight incubation at 4 °C. On the next day, 60 µl of protein A agarose was introduced to the lysate, followed by a 4-hour incubation at 4 °C. The agarose complexes containing antibodies and target proteins underwent three washes for 5 min each at 4 °C. Immunoprecipitation (IP) and Co-IP were conducted following the Western blotting protocol mentioned earlier. The resulting complex was sent to the protein mass spectrometry laboratory for IP-MS analyses using a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Scientific, Waltham). Raw data from the mass spectrometer were preprocessed with Mascot Distiller 2.4 for peak picking, and the resulting peak lists were searched against the UniProt mouse database using the Mascot 2.5 search engine.

RIP and RIP–qPCR

As previously described, RIP was conducted using 14 dpp mouse testes [24, 47]. The testes were lysed in cell lysis buffer (P0013, Beyotime, Shanghai, China), supplemented with PMSF (1:100, ST506, Beyotime, Shanghai, China), a proteinase inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China), DTT (1:50, ST041-2 ml, Beyotime, Shanghai, China), and an RNase inhibitor (1:20, R0102-10 kU, Beyotime, Shanghai, China). Following a 20-minute incubation on ice, the lysate was combined with 20 µl of protein A agarose (P2051-2 ml, Beyotime, Shanghai, China) for pre-clearing at 4 °C for 1 h. Subsequently, 2 micrograms of a BCAS2 antibody (PA2466, Abmart, Shanghai, China) and normal rabbit IgG (A7016, Beyotime, Shanghai, China) were introduced to the lysate, followed by an overnight incubation at 4 °C. On the subsequent day, 60 µl of protein A agarose was added to the lysate, and the mixture was incubated at 4 °C for 4 h. The agarose complexes, encompassing antibodies, target proteins, and RNA, underwent a 5-minute wash at 4 °C, repeated three times. Protein-bound RNA was extracted using RNAiso Plus and the Direct-zol RNA MicroPrep kit. RIP–qPCR analysis was performed following the aforementioned RT–qPCR protocol.

irCLIP-seq

Seminiferous tubules were isolated from adult WT C57BL/6 N mice testes, and ultraviolet light (254 nm) was employed to cross-link the tubules, ensuring the covalent binding of RNA-binding proteins (RBPs) to their cognate RNAs. Following this, BCAS2 and the cross-linked RNAs were subjected to immunoprecipitation with an anti-BCAS2 antibody and subsequently digested with Micrococcal nuclease (catalog no. EN0181, Thermo Fisher Scientific). An IR800-biotin adapter was ligated to the 3′ ends of the RNA fragments. The BCAS2/RNA complexes, ranging from approximately 47 to 62 kDa, were separated by SDS-PAGE gel and transferred to a nitrocellulose membrane (catalog no. HATF00010, Millipore). Subsequently, RNA and protein complexes were extracted from the nitrocellulose membrane and subjected to proteinase K digestion (catalog no. 9034, TaKaRa). RNAs were isolated using saturated phenol (catalog no. AM9712, Ambion), ligated with adaptors, and converted to cDNAs using the SuperScript III First-Strand Kit (catalog no. 18080-051, Invitrogen). The cDNAs were then amplified by PCR to generate the respective libraries, followed by sequencing on the HiSeq 2500 Illumina platform.

For the analysis of CLIP-seq data, reads were initially trimmed of adaptor sequences using Trimmomatic (version 0.36). Subsequently, bowtie 2 (version 2.1.0) was utilized to map clean reads to the mm10 reference genome with the following parameters: “-p 10 -L 15 -N 1 -D 50 -R 50 --phred33 --qc-filter --very-sensitive --end-to-end.” CLIP-seq peaks were identified using Piranha (version 1.2.1) with the parameters: “-s -b 20 -d Zero Truncated Negative Binomial -p 0.05.”

Homer (version 4.9.1) was employed for peak annotation based on the mm10 genome assembly and for the analysis of BCAS2-binding motifs. The quality of replicates was assessed by calculating the pairwise Spearman correlation coefficient.

Minigene reporter assay

Minigene reporter assay was performed as described previously [24, 48]. The WT Trp53bp1 minigene was constructed by inserting an additional 3871 bp from exons 8, 9, and 10 (corresponding to chr2: 121,248,163 − 121,252,027 mm10) into pcDNA3.0 vector between the Asc I and Pac I sites. The plasmids containing the mutant-binding site of Bcas2 were cloned with appropriate primers adjacent to the binding site using the site-directed mutagenesis approach. To generate the expression plasmid for Bcas2, the CDS region of Bcas2 was amplified and cloned into the pcDNA3.0 vector with Asc I and Pac I sites. To generate the BCAS2 knockdown vector, the designed sequence for producing BCAS2 short hairpin RNA was amplified and cloned into pMKO.1 vector with Age I and Eco RI sites. 293T cells were transfected with the BCAS2-expressing or knockdown plasmid together with indicated minigene plasmids using Lipo8000™ (C0533-1.5 ml, Beyotime, Shanghai, China) following the provided instructions. The transfected cells were harvested for RNA and protein analyses 24 h later. RT–PCR was performed with primers amplifying endogenous transcripts.

Statistical analysis

Pearson’s correlation coefficients (R) were calculated utilizing RNA-seq, CLIP-seq, and IP-MS data. To compare the distributions of CLIP-seq signals for two sets of genes, the Kolmogorov–Smirnov test was employed. Statistical analyses were conducted using GraphPad Prism software (version 9.0.0), and the presented values and error bars represent the mean ± SEM. Significant differences between the two groups were assessed using Student’s t-test, with statistical significance considered as follows: Exact P value P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.