Abstract
Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTH-null mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.
Highlights
Gonadotropin-regulated testicular RNA helicase (GRTH/ DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis
The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 were increased significantly in the GRTH-null mice. miR469 silencing of Transition protein 2 (TP2) and Protamine 2 (Prm2) mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm
Differential Expression of miRNAs from Round Spermatids in Wild Type and GRTH Knock-out Mice—miRNA expression profiles for round spermatids of wild type and GRTH knockout mice were determined by Rodent TaqMan Low Density miRNA Arrays A version 2.0 (TLDA, Applied Biosystem). miRNAs were considered to have significant differential expression if they were up- or down-regulated at least 4-fold
Summary
Gonadotropin-regulated testicular RNA helicase (GRTH/ DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Differential Expression of pri-miRNAs and Drosha-DGCR8 in Round Spermatids between Wild Type and GRTH Knock-out Mice—To elucidate the processing mechanism leading to the up-regulation of miRNAs, presented, pri-miRNAs corresponding to selected up-regulated and unchanged (control) miRNAs from the different groups were analyzed in round spermatids of GRTH-KO and WT mice by the qRT-PCR with specific gene primers located upstream to the hairpin of each precursor (Fig. 2).
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