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Gregory M. Buchold, Edward M. Eddy, Testicular Expression of Multiple DExD/H RNA Helicases., Biology of Reproduction, Volume 87, Issue Suppl_1, 1 August 2012, Page 430, https://doi.org/10.1093/biolreprod/87.s1.430
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Translational control in post-meiotic male germ cells is critical for terminal differentiation without new transcription. Nuage granules are believed important players in this process and contain multiple confirmed and additional presumed members of the Argonaute, Tudor, and DExD/H helicase families. Several DExD/H RNA helicases are components of, and genetically required for, nuage functions in invertebrates. Mammalian DExD/H helicases have a variety of roles in RNA metabolism including rRNA maturation, mRNA transport, and translational control. Gene knockouts studies show that Ddx4 (previously Mvh, mouse vasa homolog) and Ddx25 are required for male mouse fertility and are known to be associated with nuage. Ddx1 is expressed in PGCs, supports growth of germ cell tumors in nude mice, and its Drosophila ortholog belle is a nuage protein essential for male fertility. Many of the sixty members of this protein family are expressed in the testis, and affinity purification studies suggest several others are associated with PIWI complexes. To determine whether additional helicases are present in nuage granules we characterized the expression of thirty members of this family by qPCR and western blot analysis in twelve adult mouse tissues and by immunohistochemistry in the testis. Six were testis-abundant at the RNA level. None of these were testis-specific at the protein level, although two helicases were only expressed in one additional adult tissue. Similar to DDX25, other helicases were detected by immunohistochemistry in Leydig cells in addition to germ cells, while among the candidates assayed, only DDX19B was detected solely in Leydig cells in the testis. In addition, DDX39 was localized exclusively to germ cell nuclei up to spermatid nuclear condensation, while DDX21 was detected in the nucleus of preleptotene to zygotene spermatocytes but was cytoplasmic from pachytene spermatocytes to step 9 spermatids. Although two isoforms of DDX21 were observed, cytoplasmic localization was correlated with increased relative abundance of the larger form on western blot analysis. DHX30 and DHX34 were localized to the cytoplasm and present predominantly in spermatocytes and condensing spermatids, respectively. Similar to the nuage scaffold protein GASZ, DHX30 and DHX34 helicases do not have invertebrate orthologs but are strongly conserved in the vertebrate lineage, suggesting an important role in mammalian fertility. From this systematic survey of DExD/H helicases we have identified two additional nuage-associated helicases, DHX30 and DHX34. However, others might contribute to RNA regulation in the testis outside of nuage granules. This research was supported by the Intramural Research Program of the NIH, National Institutes of Environmental Health Sciences.
