ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus

• Published in: Biology of reproduction 2023-09, Vol.109 (3), p.340-355
• Main Authors: Potgieter, Sarah, Eddy, Christopher, Badrinath, Aditi, Chukrallah, Lauren, Lo, Toby, Mohanty, Gayatri, Visconti, Pablo E., Snyder, Elizabeth M.
• Format: Article
• Language: English
• Subjects: Animals; Carrier Proteins - metabolism; Cytoskeleton; Editor's Choice; Germ cells; Immunofluorescence; Infertility; LINC complex; Localization; Male; manchette; Meiosis; Mice; microtubules; Microtubules - metabolism; Mutants; Nuclear Pore - genetics; Nuclear Pore - metabolism; nuclear pore complex; Nuclear pores; Nuclear transport; Phenotypes; Protein transport; Proteins; Proteins - metabolism; RESEARCH ARTICLE; Ribonucleic acid; RNA; RNA-binding protein; RNA-binding proteins; Semen - metabolism; Sperm; Spermatids; Spermatids - metabolism; Spermatogenesis - genetics; Spermatozoa - metabolism; spermiogenesis; Testes; Testis - metabolism; Transcriptomes; Translation; translation regulation
• ISSN: 0006-3363; 1529-7268; 1529-7268
• DOI: 10.1093/biolre/ioad069

ADAD1 is a testis-specific RNA-binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm showed defective DNA compaction, abnormal head shaping, and reduced motility. Mutant testes demonstrated minimal transcriptome changes; however, ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Further, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Additional analyses demonstrated impaired subcellular localization of multiple proteins, suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC (linker of nucleoskeleton and cytoskeleton) complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1's impact on the NPC (nuclear pore complex), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1's impact on translation is required for a NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1's influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype. Summary Sentence: ADAD1 is a post-meiotic spermatid RNA-binding protein that is required for normal translation of mRNAs important for post-meiotic differentiation and mRNAs associated with nuclear and intracellular transport. Graphical Abstract