Biol Reprod. 2023 Jul 03. pii: ioad069. [Epub ahead of print]
ADAD1 is a testis-specific RNA binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm morphology and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm demonstrated defective DNA compaction, abnormal head shaping, and reduced motility. RNA sequencing of Adad1 mutant testes revealed minimal transcriptome changes however ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Additionally, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Further 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 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 nuclear pore complex (NPC), 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 is required for normal translation of the 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.
Keywords: LINC complex; RNA-binding proteins; manchette; microtubules; nuclear pore complex; protein transport; spermatids; spermiogenesis; translation regulation