Fig 2: Ranbp9 deficiency causes male germ cell apoptosis and DNA double-strand breaks.(A) TUNEL assays on WT, Ranbp9 global KO (Ranbp9Δ/Δ) and gcKO testes. Arrows point to apoptotic cells stained in brown. Scale bar = 50 µm. Significantly increased average number of apoptotic cells is observed in both Ranbp9 Δ/Δ and gcKO testis (the far right panel). >60 cross-sections were scored for the average number of apoptotic cells per tubule for each genotype. Three mice of each genotype were analyzed, and data were presented as mean ± SD, n = 3. (B) Immunofluorescence staining of γH2AX in seminiferous tubules of WT and gcKO testes at ∼stage VI. In WT seminiferous tubules, γH2AX immunoreactivity is mostly confined to the XY body (arrows) in pachytene spermatocytes and completely absent in round spermatids (arrowheads). In contrast, in gcKO seminiferous tubules, numerous round spermatids exhibit strong γH2AX staining (arrowheads) in addition to its normal localization in the XY body (arrow) in pachytene spermatocytes. (C) qPCR analyses showing significantly reduced levels of Prm1, Prm2, Tnp1 and Tnp2 mRNAs in 6-week old Ranbp9 gcKO testes. Data are presented as mean ± SEM, n = 3.

Fig 3: Expression profiles of Ranbp9 during testicular development and spermatogenesis in mice.(A) qPCR analyses of Ranbp9 mRNA levels in multiple organs in mice. Data are presented as mean ± SEM, n = 3. (B) Expression of Ranbp9 and Ranbp10 during postnatal testicular development. Levels of Ranbp9 and Ranbp10 mRNAs in developing testes at postnatal day 7 (P7), P14, P21, P28, P35, and in adult (Ad) were analyzed using qPCR. Data are presented as mean ± SEM, n = 3. (C) Expression of RANBP9 protein during postnatal testicular development. Levels of RANBP9 in the testes from newborn (P0), postnatal day 3 (P3), P7, P14, P21, P28, and P35 and adult male mice were determined using western blot analyses. ACTIN was used as a loading control. (D) Immunofluorescent detection of RANBP9 in homozygous Ranbp9 flox (Ranbp9lox/lox) and Ranbp9 global knockout (Ranbp9Δ/Δ) testes. In Ranbp9lox/lox testes, RANBP9 immunoreactivity was mostly detected in the nucleus of spermatocytes (spc) and spermatids (spd). Insets show the digitally magnified view of the framed area. RANBP9 was also detected in the nucleolus of Sertoli cells (Ser), and in both the cytoplasm and the nucleus in interstitial Leydig cells (Ley) (Middle panels). While the nucleus was partially RANBP9-positive in a subpopulation of spermatogonia (spg), RANBP9 staining covered the entire nucleus in both pachytene spermatocytes (pachy) and round spermatids (rspd) (Lower panels). In Ranbp9Δ/Δ testes, RANBP9 staining was completely absent. Scale bar = 50 µm.

Fig 4: Disruptions of the mRNA transcriptome and alternative splicing patterns in Ranbp9 gcKO testes.(A) Scatter plot showing significantly de-regulated transcripts in Ranbp9 gcKO testes compared to WT controls. Blue dots (2,313) represent significantly upregulated transcripts, while red dots (316) denote significantly downregulated transcripts (p<0.05, fold change>2). Yellow dots illustrate unchanged transcripts. (B) Venn diagram showing the number of unique transcript isoforms detected in Ranbp9 gcKO (2,420) and WT (277) testes. (C) Distribution of 1,816 aberrant splicing events (insertions or deletions) along the entire length of mRNAs in gcKO testes. The y-axis represents the size of insertions (positive values) or deletions (negative values), whereas the x-axis denotes location percentage (splicing location/total transcript size), reflecting the relative position of splicing events along the entire length of the transcripts, e.g., 0% refers to the very 3′end, 50% means the middle of the transcript and 100% indicates the very 5′end. (D) Semi-qPCR-based detection of aberrant alterative splicing patterns in three RANBP9 direct target mRNAs (Rfx2, Plec and Usp19). Lower panels represent the schematic diagram of alternatively spliced exons detected by RNA-Seq analysis. Gapdh was used as a loading control.

Fig 5: RANBP9 binds numerous mRNAs and affects their expression levels at least partially through affecting alternative splicing.(A) A representative mRNA assembly output showing RIP-Seq reads for Ddx25 identified from the RIP products using the RANBP9 antibody and IgG (control). (B) qPCR analyses of levels of three RANBP9-bound mRNAs (Rfx2, Plec and Usp19) in WT and gcKO testes. All three are highly enriched in WT compared to gcKO testes, demonstrating the specificity of the anti-RANBP9 antibody used in RIP-Seq assays. (C) GO enrichment analyses of RANBP9-bound mRNAs identified using RIP-Seq. (D) Venn diagram showing the number of up- and down-regulated RANBP9-bound target transcripts in gcKO testes (P<0.05, fold change>1.5). (E) qPCR analyses of levels of 17 RANBP9 target transcripts in 6-week-old WT and gcKO testes. Data are presented as mean ± SEM, and significantly altered levels were marked with * (n = 4, P<0.05).