Fig 2: Proteins for sperm flagellum development are down-regulated in KO mice. a The heat map from three independent proteomic analyses of testes from the WT and KO mice. b The proteins down-regulated in the KO mice are associated with sperm flagellum development and related energy metabolism. c The different MS/MS spectra of Akap3, Odf2, Tssk4, March10, Ropn1, and Cabyr in the KO mice and the WT mice. d The expression of the key proteins in the testes of the WT and KO mice. The western blotting results confirmed the decreased expression of Akap3, Odf2, Tssk4, March10, Ropn1 and Cabyr in the KO mice compared with the WT mice. (n = 4 biologically independent WT mice or KO mice). e The location of the vital proteins in human spermatozoa by immunofluorescence staining. AKAP3 (green), ODF2 (green), TSSK4 (green), MARCH10 (green), CABYR (green), and ROPN1 (red) are dominantly expressed in human spermatozoa flagella. (blue, DAPI; scale bars, 5 µm)

Fig 3: The mechanisms of QRICH2 regulating sperm flagellar formation. a The mRNA levels of Akap3, Odf2, Tssk4, March10, Cabyr and Ropn1 in the testes of the KO and WT mice. The reduced mRNA levels of Odf2 and Cabyr in the KO mice were detected by real-time PCR (Student’s t-test; n = 3 independent experiments; *p < 0.05; NS, not significant; error bars, s.e.m). b One QRICH2-occupied site in the ODF2 promoter and two QRICH2-occupied sites in the CABYR promoter were detected by ChIP-PCR assays of human testicular tissues. Input DNA and normal mouse IgG pulldowns were used as positive control and negative control, respectively. c ChIP-qPCR results showed the increased binding of QRICH2 to the ODF2 and CABYR promoter regions in the group with QRICH2 antibody compared to that with IgG (Student’s t-test; n = 3 independent experiments; *p < 0.05; error bars, s.e.m). d QRICH2 could enhance the transcriptional activity of ODF2 and CABYR by luciferase reporter assays (Student’s t-test; n = 3 independent experiments; *p < 0.05; error bars, s.e.m). e The increased ubiquitination levels of Akap3, Tssk4, Ropn1 and March10 in the testes of the KO mice compared with that in the WT mice. f, g QRICH2 decreased the ubiquitylation of exogenous AKAP3 (f) and TSSK4 (g) in HEK 293 cells. The cells were harvested after a 6-h treatment with 10 µM of MG132. h The binding of Ropn1 and Cabyr to Akap3 was weakened in the testes of the KO mice compared with that in the WT mice. i Overexpression of TSSK4 could increase the protein levels of ODF2