Testis-specific miRNA-469 Up-regulated in Gonadotropin-regulated Testicular RNA Helicase (GRTH/DDX25)-null Mice Silences Transition Protein 2 and Protamine 2 Messages at Sites within Coding Region
2011; Elsevier BV; Volume: 286; Issue: 52 Linguagem: Inglês
10.1074/jbc.m111.282756
ISSN1083-351X
AutoresLisheng Dai, Chon‐Hwa Tsai‐Morris, Hisashi Satô, Joaquín Villar, Jung‐Hoon Kang, Jiabao Zhang, Maria Dufau,
Tópico(s)RNA Research and Splicing
ResumoGonadotropin-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. 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. IntroductionMammalian spermatogenesis is a complex process in which primary germ cells undergo mitotic and meiotic divisions to generate haploid round spermatids, and proceed to the differentiation process of spermiogenesis that produces elongating spermatids and mature sperm. This process is regulated at the transcriptional and post-transcriptional levels by the integrated expression of an array of testicular genes in a precise temporal sequence (1Steger K. Anat. Embryol. 2001; 203: 323-334Crossref PubMed Scopus (121) Google Scholar, 2Eddy E.M. Recent Prog. Horm. Res. 2002; 57: 103-128Crossref PubMed Scopus (341) Google Scholar). Chromatin compactation that occurs in elongated spermatids during spermiogenesis is essential for nuclear condensation to generate mature spermatozoa. This repackaging event is achieved by replacing histones with transition proteins (TP1 and TP2), which in turn are replaced by protamines (Prm1 and Prm2). The initial active transcription phase with translational repression is followed by cessation of transcription associated with chromatin modifications. mRNA of genes that are essential for later stages of spermiogenesis are generated well prior their translation. Several mRNAs that associate with messenger ribonuclear proteins are repressed translationally at cytoplasmic sites, presumably in the chromatoid body of round spermatids.Gonadotropin-regulated testicular RNA helicase (GRTH 2The abbreviations used are: GRTHgonadotropin-regulated testicular RNA helicasentnucleotideqRTquantitative RTmiRNAmicroRNACBchromatoid bodyMREmiR-469 response elementCDcoding regionpri-miRNAprimary miRNA. /DDX25), a testis-specific member of the DEAD (Asp-Glu-Gly-Asp)-box family present in Leydig and germ cells (meiotic spermatocytes and round spermatids) is regulated developmentally by androgen at the transcriptional level (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. J. Biol. Chem. 1999; 274: 37932-37940Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Sheng Y. Tsai-Morris C.H. Dufau M.L. J. Biol. Chem. 2003; 278: 27796-27803Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Dufau M.L. Tsai-Morris C.H. Trends Endocrinol. Metab. 2007; 18: 314-320Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 6Tsai-Morris C.H. Sheng Y. Gutti R.K. Tang P.Z. Dufau M.L. J. Androl. 2010; 31: 45-52Crossref PubMed Scopus (28) Google Scholar). GRTH is a multifunctional protein, and as component of messenger ribonuclear protein, it transports target mRNAs from the nucleus to cytoplasmic sites (chromatoid bodies, a perinuclear organelle of nuage structure in spermatids for storage/processing) and to polyribosomes for translation (7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). GRTH is essential to govern the structure of the chromatoid body (CB) and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis (8Sato H. Tsai-Morris C.H. Dufau M.L. Biochim. Biophys. Acta. 2010; 1803: 534-543Crossref PubMed Scopus (26) Google Scholar). GRTH knock-out male mice are sterile, with azoospermia caused by a complete arrest of spermiogenesis at step 8/9 of round spermatids and failure to elongate. The CB was condensed and greatly reduced in size in the round spermatids of GRTH-null mice (9Tsai-Morris C.H. Sheng Y. Lee E. Lei K.J. Dufau M.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6373-6378Crossref PubMed Scopus (132) Google Scholar). Accumulated evidence indicates that members of the small interfering RNA (siRNA) and microRNA (miRNA) pathway, which include Dicer to process si/miRNA precursors to mature small RNAs, the effector complex RNA-induced silencing complex, and mouse vasa homolog, another germ cell helicase, are present in the CB (10Kotaja N. Bhattacharyya S.N. Jaskiewicz L. Kimmins S. Parvinen M. Filipowicz W. Sassone-Corsi P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2647-2652Crossref PubMed Scopus (283) Google Scholar, 11Kotaja N. Sassone-Corsi P. Nat. Rev. Mol. Cell Biol. 2007; 8: 85-90Crossref PubMed Scopus (233) Google Scholar). Preservation of the expression of relevant mRNAs with failure of protein expression such as angiotensin-converting enzyme, transition protein 1 (TP1), and transition protein 2 (TP2) in the GRTH-null mice indicate that GRTH is also involved in post-transcriptional events.microRNAs, a class of ∼22-nt noncoding RNAs, participate in diverse biological functions by promoting degradation and inhibition of translation of target mRNAs. With the exception of miRNAs generated within introns of protein-coding genes, most miRNAs are derived from primary miRNA transcripts (pri-miRNAs) transcribed by RNA polymerase II, which are 5′-capped and polyadenylated (12Han J. Lee Y. Yeom K.H. Kim Y.K. Jin H. Kim V.N. Genes Dev. 2004; 18: 3016-3027Crossref PubMed Scopus (1544) Google Scholar, 13Han J. Lee Y. Yeom K.H. Nam J.W. Heo I. Rhee J.K. Sohn S.Y. Cho Y. Zhang B.T. Kim V.N. Cell. 2006; 125: 887-901Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar). pri-miRNAs are subsequently processed by the Drosha-DGCR8 complex in the nucleus to generate precursor miRNAs (pre-miRNAs), which are exported by Exportin-5 to the cytoplasm where mature miRNAs are generated via a Dicer-dependent or -independent route (14Krol J. Loedige I. Filipowicz W. Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3452) Google Scholar, 15Miyoshi K. Miyoshi T. Siomi H. Mol. Genet. Genomics. 2010; 284: 95-103Crossref PubMed Scopus (158) Google Scholar). Recent findings of individual miRNAs expressed during spermatogenesis in a developmental stage-specific manner suggest the participation of miRNAs in male germ cell development through their contribution to cell type-specific profiles of protein expression during spermatogenesis (16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar, 17Ro S. Park C. Sanders K.M. McCarrey J.R. Yan W. Dev. Biol. 2007; 311: 592-602Crossref PubMed Scopus (202) Google Scholar).To elucidate regulatory actions of GRTH in miRNA processing during germ cell development, we first compared the differential expression profiles of miRNA in purified round spermatids from wild type and GRTH null mice. A panel of miRNAs and some of their corresponding pri-miRNAs was significantly increased in the GRTH-null mice. Among the various up-regulated miRNAs, the testis-specific miR-469 with cell expression similar to GRTH was selected to pursue further studies (16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar). miR-469 repressed the translation of TP2 and Prm2 through binding to the coding region of TP2 and Prm2 mRNAs. This is consistent with the preservation of TP2 and Prm2 mRNA with the failure of their protein expression found in the GRTH null mice (7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Also, GRTH appears to negatively regulate Drosha/DGCR8 gene expression to control microRNA maturation. This study has provided insights into a novel molecular control mechanism of GRTH through the microRNA pathway in the regulation of spermatogenesis.DISCUSSIONThis study has demonstrated a subset of microRNAs that were differentially up-regulated in round spermatids of GRTH KO mice, including testis-specific miR-469, testis-preferred (miR-34c and miR-470), ubiquitous let-7 family members, and others that might participate in germ cell development (Fig. 1). The primary miRNAs of interest were also increased along with the enzyme complex (Drosha-DGCR8) that promotes the processing of primary miRNA transcripts. Our studies provide evidence that miR-469 specifically targets chromatin remodeling gene products TP2 and Prm2, which are essential for germ cell development, and further understanding of the GRTH regulatory function during spermatogenesis.It is interesting to note in the present microarray analysis the presence of solely differentially up-regulated microRNAs (Fig. 1, I and II) covering a wide range cellular actions that could participate in germ cell development. This type of up-regulation was similarly observed in the absence of Tdrd6 gene expression where the majority of differentially expressed miRNAs (91%) were up-regulated. Tdrd6 is a Tudor domain containing protein that exhibits the same pattern of expression as GRTH and is also required for the architecture of the chromatoid and the progress of spermiogenesis (9Tsai-Morris C.H. Sheng Y. Lee E. Lei K.J. Dufau M.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6373-6378Crossref PubMed Scopus (132) Google Scholar, 22Vasileva A. Tiedau D. Firooznia A. Müller-Reichert T. Jessberger R. Curr. Biol. 2009; 19: 630-639Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Only the miRNAs miR-29c, miR-34a, miR-34c, and miR-470 were commonly up-regulated in both null mice models. Target scan predicted DNA-modifying enzymes (DNA methyltransferase and histone deacetylase), RNA-processing enzyme (Dicer), and eukaryotic translational initiation factors as the miRNA target genes. This suggests a common control mechanism that might be shared by both GRTH and Tdrd6. Some aspects of the specificity of GRTH action differing from Tdrd6 rely on the unique regulation of miRNAs like miR-469 through its association with the coding region of chromatin remodeling genes TP2 and Prm2 (FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6). Other not yet identified miRNAs differentially regulated by GRTH could also target TP2 and Prm2 in this mouse model. Neither target scan nor RNA22 program revealed any other miRNA candidates in the panel of differentially expressed species (WT versus KO) targeting TP2 and Prm2 in this study. The interaction between miRNAs and target genes seems to play an important factor in the regulation of stage-specific expression of germ cell genes.In addition to the fact that both GRTH and miR-469 are expressed specifically in the testis, the striking similarity of their increased expression from puberty to adulthood (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. J. Biol. Chem. 1999; 274: 37932-37940Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar), and the cell-specific expression in the late stage of meiosis and round spermatids (4Sheng Y. Tsai-Morris C.H. Dufau M.L. J. Biol. Chem. 2003; 278: 27796-27803Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar, 17Ro S. Park C. Sanders K.M. McCarrey J.R. Yan W. Dev. Biol. 2007; 311: 592-602Crossref PubMed Scopus (202) Google Scholar) indicate their functional importance during testicular spermatogenesis. Evidence for the presence of mature miR-469 in round spermatids and its functional role in the regulation of genes (TP2/Prm2) required for chromatin remodeling during spermatogenesis have been shown in this study. The finding of an increased miR-469 level in the absence of GRTH (Fig. 1) led us to propose that GRTH might have an intrinsic regulatory role to prevent overexpression of miR-469 and possible others shown to be up-regulated in this study.miR-34c, is also known to be highly expressed in the spermatocytes and round spermatids (23Bouhallier F. Allioli N. Lavial F. Chalmel F. Perrard M.H. Durand P. Samarut J. Pain B. Rouault J.P. RNA. 2010; 16: 720-731Crossref PubMed Scopus (210) Google Scholar). Although the RNA22 program predicted few potential low energy binding sites in the target genes of interest (Fig. 3) governed by GRTH (7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), none of these genes were proven as miR-34c targets by the dual-luciferase analysis (data not shown). However, it is conceivable that the miR-34c target Transforming growth factor inhibitor 2 (TGIF2), a known inhibitor of the TGFβ receptor/signaling pathway for its regulation during spermatogenesis (24Damestoy A. Perrard M.H. Vigier M. Sabido O. Durand P. Reprod. Biol. Endocrinol. 2005; 3: 22Crossref PubMed Scopus (26) Google Scholar, 25Itman C. Loveland K.L. Dev. Dyn. 2008; 237: 97-111Crossref PubMed Scopus (66) Google Scholar), might be linked to GRTH regulation. Other evolutionarily conserved members of miR-34 family, miR-34a and -34b, known to have a role in determining cell fate, provide a new avenue for the GRTH action in the germ cells.From the 13 let-7 miRNAs family members evolutionarily conserved from Drosophila to human, let-7a/d, -b, and -e–g were up-regulated in GRTH KO mice. let-7 members have either activator or repressor function depending on specific cellular stages. let-7 is known to target endonuclease Dicer within its coding region as a mechanism for a miRNA/Dicer autoregulatory negative feedback loop (26Forman J.J. Legesse-Miller A. Coller H.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 14879-14884Crossref PubMed Scopus (455) Google Scholar). This appears not to be the case in the GRTH KO mice model where no change in Dicer protein expression was observed (8Sato H. Tsai-Morris C.H. Dufau M.L. Biochim. Biophys. Acta. 2010; 1803: 534-543Crossref PubMed Scopus (26) Google Scholar) despite the marked increases noted in several members of the let-7 family (Fig. 1I, panel B). High level of let-7 expression has been proposed as a unique property of testis-derived male stem cell germ line for primordial germ cells' proliferation and development (27Jung Y.H. Gupta M.K. Shin J.Y. Uhm S.J. Lee H.T. Mol. Hum. Reprod. 2010; 16: 804-810Crossref PubMed Scopus (35) Google Scholar, 28Hayashi K. Chuva de Sousa Lopes S.M. Kaneda M. Tang F. Hajkova P. Lao K. O'Carroll D. Das P.P. Tarakhovsky A. Miska E.A. Surani M.A. PLoS One. 2008; 3e1738 Crossref PubMed Scopus (387) Google Scholar, 29West J.A. Viswanathan S.R. Yabuuchi A. Cunniff K. Takeuchi A. Park I.H. Sero J.E. Zhu H. Perez-Atayde A. Frazier A.L. Surani M.A. Daley G.Q. Nature. 2009; 460: 909-913Crossref PubMed Scopus (314) Google Scholar). In contrast, in many human cancers, let-7 expression is deregulated, and let-7 is widely viewed as a tumor suppressor (30Boyerinas B. Park S.M. Hau A. Murmann A.E. Peter M.E. Endocr. Relat. Cancer. 2010; 17: F19-F36Crossref PubMed Scopus (556) Google Scholar). In pituitary adenomas, the negative regulation of high mobility group protein A2 (HMGA2) by let-7 is believed to contribute to the pathogenesis of the disease (31Qian Z.R. Asa S.L. Siomi H. Siomi M.C. Yoshimoto K. Yamada S. Wang E.L. Rahman M.M. Inoue H. Itakura M. Kudo E. Sano T. Mod. Pathol. 2009; 22: 431-441Crossref PubMed Scopus (112) Google Scholar). The HMGA2 is a non-histone chromatin protein that binds DNA in AT regions and can regulate transcription by altering chromatin structure and consequently promoting the assembly of transcription factor complexes (32Sgarra R. Rustighi A. Tessari M.A. Di Bernardo J. Altamura S. Fusco A. Manfioletti G. Giancotti V. FEBS Lett. 2004; 574: 1-8Crossref PubMed Scopus (188) Google Scholar). Its expression is highest in pachytene spermatocytes and early spermatids, and disruption of the HMGA2 gene results in a block of spermatogenesis with testis showing degenerating spermatocytes and few spermatids (33Chieffi P. Battista S. Barchi M. Di Agostino S. Pierantoni G.M. Fedele M. Chiariotti L. Tramontano D. Fusco A. Oncogene. 2002; 21: 3644-3650Crossref PubMed Scopus (87) Google Scholar). In the GRTH KO mice, the HMGA2 protein is completely absent, but its mRNA level was unchanged compared with the WT (7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). let-7 increases in the GRTH KO could effectively silence the HMGA2 mRNA and prevent its expression, and its deficiency could contribute to the phenotype observed in the GRTH KO mice. Thus, let-7 regulation of germ cell target induced by GRTH regulation during spermatogenesis could be envisioned.Identification of putative functional response element (MRE) sites localized only within the coding region of TP2 and Prm2 provides evidence for a nonclassical regulation of these target genes by miR-469 (FIGURE 4, FIGURE 5, FIGURE 6). It was further demonstrated that the regulation occurred through the repression of protein translation with no effect on mRNA degradation (Fig. 7). Most of the reported functional mammalian microRNAs were found to target 3′-UTR sequences, and few studies have revealed 5′-UTR participation. More recently, evidence of elements within the coding region of mRNA have been found to be targeted by miRNAs (34Lee E.K. Gorospe M. RNA Biol. 2011; 8: 44-48Crossref PubMed Scopus (31) Google Scholar). Our studies have clearly shown exclusive participation of functional binding sites in the coding region and excluded cooperating 3′-UTR sequences for miR-469. The presence of functional binding sites for both genes was demonstrated in their mRNA coding region (two for Prm2 and one for TP2) through assessment by deletions and mutagenesis. The RNA folding program further confirmed those functional motifs by their complete complementarity with miR-469 5′ seed sequence (at bp 2–8 of the microRNA) to form stable RNA-miRNA duplex with low energy (FIGURE 4, FIGURE 5, FIGURE 6) (35Brennecke J. Stark A. Russell R.B. Cohen S.M. PLoS Biol. 2005; 3: e85Crossref PubMed Scopus (1828) Google Scholar). The functional importance of miRNA targeting coding regions and mechanism of miRNA-mediated translational repression are not well understood compared with the 3′-UTR (14Krol J. Loedige I. Filipowicz W. Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3452) Google Scholar). We speculate that the presence of two functional miR-469 MREs in Prm2 versus one in TP2 might contribute to the higher degree of down-regulation in the protein level of Prm2 compared with TP2 observed in the dual-luciferase assay (Fig. 3) and in vitro expression studies (Fig. 7).Progress has been gradually unveiled in understanding the function of miRNAs during male germ cell development. The base pairing complementarity of the microRNA with imperfect duplexes guides the endoribonuclease Argonaute (Ago) to repress the translational level or alternatively to degrade the target mRNA directly (36Shyu A.B. Wilkinson M.F. van Hoof A. EMBO J. 2008; 27: 471-481Crossref PubMed Scopus (356) Google Scholar). Previous evidence relates to the degradation of target gene mRNA message through mRNA deadenylation, such as down-regulation of TP2 by miR-122a (16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar), TGIF2, and NOTCH2 by miR-34c (23Bouhallier F. Allioli N. Lavial F. Chalmel F. Perrard M.H. Durand P. Samarut J. Pain B. Rouault J.P. RNA. 2010; 16: 720-731Crossref PubMed Scopus (210) Google Scholar), and heat shock factor 2 (HSF2) by miR-18 (37Björk J.K. Sandqvist A. Elsing A.N. Kotaja N. Sistonen L. Development. 2010; 137: 3177-3184Crossref PubMed Scopus (96) Google Scholar). miR-122a reported to specifically associate with the 3′-UTR of TP2 (16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar) was not identified in the present differential microarray panel (GRTH WT versus KO). In the case of miR-469-mediated down-regulation of Prm2 and TP2, our studies point to the repression of both Prm2 and TP2 at the translational level with minor effect on mRNA degradation. This is in contrast to a recent study indicating the predominant destabilization of targets with at least one 3′-UTR site by microRNAs (38Guo H. Ingolia N.T. Weissman J.S. Bartel D.P. Nature. 2010; 466: 835-840Crossref PubMed Scopus (3060) Google Scholar). In our case, two target sites for miR-469 in each Prm2 and TP2 mRNA were located within their coding region (FIGURE 4, FIGURE 5, FIGURE 6).Gene expression such as Prm2 and TP2 in haploid spermatids requires temporal uncoupling of transcription and translation. These mRNAs destined for translation during the late spermiogenesis phase (elongating and elongated spermatids, steps 9–16) are actively transcribed prior to the step 9 of round spermatids. It has been long postulated that those messages are temporarily stored in the chromatoid body to await the specific time to be translated (10Kotaja N. Bhattacharyya S.N. Jaskiewicz L. Kimmins S. Parvinen M. Filipowicz W. Sassone-Corsi P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2647-2652Crossref PubMed Scopus (283) Google Scholar, 11Kotaja N. Sassone-Corsi P. Nat. Rev. Mol. Cell Biol. 2007; 8: 85-90Crossref PubMed Scopus (233) Google Scholar). Translation followed by chromatin condensation and nuclear elongation ultimately progresses to the generation of mature germ cells. Expression profiling array in the mouse testis (17Ro S. Park C. Sanders K.M. McCarrey J.R. Yan W. Dev. Biol. 2007; 311: 592-602Crossref PubMed Scopus (202) Google Scholar) showed a high level of miR-469 expression coincident with the phase of classic active transcription and suppressed TP2/Prm2 translation in the meiotic and early haploid germ cells. This is followed by its decreased expression in elongating spermatids with active TP2/Prm2 protein production. The direct evidence on miR-469 targeting these messages to repress the translation observed in this study (Fig. 7) support the physiological relevance of miR-469 in germ cell development under GRTH regulation.The chromatoid body of haploid round spermatids has been viewed as the center of miRNA-mediated mRNA control to coordinate post-transcriptional gene expression during spermiogenesis (10Kotaja N. Bhattacharyya S.N. Jaskiewicz L. Kimmins S. Parvinen M. Filipowicz W. Sassone-Corsi P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2647-2652Crossref PubMed Scopus (283) Google Scholar, 11Kotaja N. Sassone-Corsi P. Nat. Rev. Mol. Cell Biol. 2007; 8: 85-90Crossref PubMed Scopus (233) Google Scholar). GRTH does not associate with any of the components of the RNA-induced silencing complex or other RNA helicase like Mvh involved in this regulation, but it is required for the preservation of the structural integrity of the CB and as transport protein to deliver mRNAs for silencing or processing (8Sato H. Tsai-Morris C.H. Dufau M.L. Biochim. Biophys. Acta. 2010; 1803: 534-543Crossref PubMed Scopus (26) Google Scholar). The up-regulated miRNAs found in the GRTH−/− mice most likely function at cytoplasmic sites other than the CB because this organelle was strikingly condensed and highly decreased in size in GRTH-null mice (9Tsai-Morris C.H. Sheng Y. Lee E. Lei K.J. Dufau M.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6373-6378Crossref PubMed Scopus (132) Google Scholar). Moreover, the relevant proteins of the RNA-induced silencing complex that normally reside in the CB of the WT mice are excluded from this organelle in the GRTH KO (8Sato H. Tsai-Morris C.H. Dufau M.L. Biochim. Biophys. Acta. 2010; 1803: 534-543Crossref PubMed Scopus (26) Google Scholar). Dual actions of GRTH in controlling at the nuclear level both primary miRNA (pri-miRNA) of interest and enzymes (Drosha and DGCR8) point to its importance in germ cell miRNA biogenesis (Fig. 2). A connection between testis-specific GRTH, Drosha/DGCR8, mature miR-469, and Prm2/TP2 has been implied indirectly from our comparative studies in GRTH wild type versus GRTH KO mice. However, direct demonstration of this microRNA biogenesis and its impact in Prm2/TP2 protein expression must await the development of suitable germ cell cultures of spermatocytes and round spermatids that would permit optimal silencing of multiple messages (i.e. Drosha/DGCR8/Dicer) and quantitative evaluation of protein expression.Because both pri-mRNA and enzyme complexes are localized in the nucleus, the 56-kDa nuclear form of GRTH protein is likely to regulate their expression at the level of transcription and/or message stability through a not yet identified mechanism. Our recent studies (not shown) have demonstrated comparable half-lives of Drosha and DGCR8 messages in GRTH wild type and GRTH KO mice. This indicates that GRTH could directly or indirectly regulate members of the microprocessor complex at the transcriptional level. Unlike members of DEAD-box protein family, p68 and p72, which associate Drosha-DGCR8 microprocessor complex (39Fukuda T. Yamagata K. Fujiyama S. Matsumoto T. Koshida I. Yoshimura K. Mihara M. Naitou M. Endoh H. Nakamura T. Akimoto C. Yamamoto Y. Katagiri T. Foulds C. Takezawa S. Kitagawa H. Takeyama K. O'Malley B.W. Kato S. Nat. Cell Biol. 2007; 9: 604-611Crossref PubMed Scopus (334) Google Scholar), GRTH does not associate with this complex as was determined by immunoprecipitation. Also, it was not associated with mRNAs of Drosha and DGCR8 in combined immunoprecipitation/RT-PCR studies (data not shown).It is conceivable that during early stages of the haploid phase (round spermatids) miR-469 represses TP2 and Prm2 protein expression by silencing message translation in these germ cells. In elongated spermatids, decreased miR-469 expression (17Ro S. Park C. Sanders K.M. McCarrey J.R. Yan W. Dev. Biol. 2007; 311: 592-602Crossref PubMed Scopus (202) Google Scholar) could release the translational repression of TP2 and Prm2 to proceed with the mRNA processing in a sequential manner. In the case of GRTH-null mice, TP2 and Prm2 mRNA translations were repressed due to the aberrant miR-469 expression level. Moreover, transport of relevant messages to polysomal sites by GRTH and its proposed function in translation is curtailed in GRTH KO mice (5Dufau M.L. Tsai-Morris C.H. Trends Endocrinol. Metab. 2007; 18: 314-320Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 6Tsai-Morris C.H. Sheng Y. Gutti R.K. Tang P.Z. Dufau M.L. J. Androl. 2010; 31: 45-52Crossref PubMed Scopus (28) Google Scholar, 7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Increased miRNAs in the GRTH-null mice could silence the protein expression of a number of genes post-transcriptionally and thus cause spermatogenesis arrest at the round spermatid stage 7–8 and failure to elongate (Fig. 8).In summary, we conclude that a negative regulation of GRTH via the route of miRNA biogenesis/action may play an important role during spermatogenesis (Fig. 8). These regulatory aspects provide insights into a novel molecular mechanism of GRTH function during male germ cell development. IntroductionMammalian spermatogenesis is a complex process in which primary germ cells undergo mitotic and meiotic divisions to generate haploid round spermatids, and proceed to the differentiation process of spermiogenesis that produces elongating spermatids and mature sperm. This process is regulated at the transcriptional and post-transcriptional levels by the integrated expression of an array of testicular genes in a precise temporal sequence (1Steger K. Anat. Embryol. 2001; 203: 323-334Crossref PubMed Scopus (121) Google Scholar, 2Eddy E.M. Recent Prog. Horm. Res. 2002; 57: 103-128Crossref PubMed Scopus (341) Google Scholar). Chromatin compactation that occurs in elongated spermatids during spermiogenesis is essential for nuclear condensation to generate mature spermatozoa. This repackaging event is achieved by replacing histones with transition proteins (TP1 and TP2), which in turn are replaced by protamines (Prm1 and Prm2). The initial active transcription phase with translational repression is followed by cessation of transcription associated with chromatin modifications. mRNA of genes that are essential for later stages of spermiogenesis are generated well prior their translation. Several mRNAs that associate with messenger ribonuclear proteins are repressed translationally at cytoplasmic sites, presumably in the chromatoid body of round spermatids.Gonadotropin-regulated testicular RNA helicase (GRTH 2The abbreviations used are: GRTHgonadotropin-regulated testicular RNA helicasentnucleotideqRTquantitative RTmiRNAmicroRNACBchromatoid bodyMREmiR-469 response elementCDcoding regionpri-miRNAprimary miRNA. /DDX25), a testis-specific member of the DEAD (Asp-Glu-Gly-Asp)-box family present in Leydig and germ cells (meiotic spermatocytes and round spermatids) is regulated developmentally by androgen at the transcriptional level (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. J. Biol. Chem. 1999; 274: 37932-37940Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Sheng Y. Tsai-Morris C.H. Dufau M.L. J. Biol. Chem. 2003; 278: 27796-27803Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Dufau M.L. Tsai-Morris C.H. Trends Endocrinol. Metab. 2007; 18: 314-320Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 6Tsai-Morris C.H. Sheng Y. Gutti R.K. Tang P.Z. Dufau M.L. J. Androl. 2010; 31: 45-52Crossref PubMed Scopus (28) Google Scholar). GRTH is a multifunctional protein, and as component of messenger ribonuclear protein, it transports target mRNAs from the nucleus to cytoplasmic sites (chromatoid bodies, a perinuclear organelle of nuage structure in spermatids for storage/processing) and to polyribosomes for translation (7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). GRTH is essential to govern the structure of the chromatoid body (CB) and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis (8Sato H. Tsai-Morris C.H. Dufau M.L. Biochim. Biophys. Acta. 2010; 1803: 534-543Crossref PubMed Scopus (26) Google Scholar). GRTH knock-out male mice are sterile, with azoospermia caused by a complete arrest of spermiogenesis at step 8/9 of round spermatids and failure to elongate. The CB was condensed and greatly reduced in size in the round spermatids of GRTH-null mice (9Tsai-Morris C.H. Sheng Y. Lee E. Lei K.J. Dufau M.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6373-6378Crossref PubMed Scopus (132) Google Scholar). Accumulated evidence indicates that members of the small interfering RNA (siRNA) and microRNA (miRNA) pathway, which include Dicer to process si/miRNA precursors to mature small RNAs, the effector complex RNA-induced silencing complex, and mouse vasa homolog, another germ cell helicase, are present in the CB (10Kotaja N. Bhattacharyya S.N. Jaskiewicz L. Kimmins S. Parvinen M. Filipowicz W. Sassone-Corsi P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2647-2652Crossref PubMed Scopus (283) Google Scholar, 11Kotaja N. Sassone-Corsi P. Nat. Rev. Mol. Cell Biol. 2007; 8: 85-90Crossref PubMed Scopus (233) Google Scholar). Preservation of the expression of relevant mRNAs with failure of protein expression such as angiotensin-converting enzyme, transition protein 1 (TP1), and transition protein 2 (TP2) in the GRTH-null mice indicate that GRTH is also involved in post-transcriptional events.microRNAs, a class of ∼22-nt noncoding RNAs, participate in diverse biological functions by promoting degradation and inhibition of translation of target mRNAs. With the exception of miRNAs generated within introns of protein-coding genes, most miRNAs are derived from primary miRNA transcripts (pri-miRNAs) transcribed by RNA polymerase II, which are 5′-capped and polyadenylated (12Han J. Lee Y. Yeom K.H. Kim Y.K. Jin H. Kim V.N. Genes Dev. 2004; 18: 3016-3027Crossref PubMed Scopus (1544) Google Scholar, 13Han J. Lee Y. Yeom K.H. Nam J.W. Heo I. Rhee J.K. Sohn S.Y. Cho Y. Zhang B.T. Kim V.N. Cell. 2006; 125: 887-901Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar). pri-miRNAs are subsequently processed by the Drosha-DGCR8 complex in the nucleus to generate precursor miRNAs (pre-miRNAs), which are exported by Exportin-5 to the cytoplasm where mature miRNAs are generated via a Dicer-dependent or -independent route (14Krol J. Loedige I. Filipowicz W. Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3452) Google Scholar, 15Miyoshi K. Miyoshi T. Siomi H. Mol. Genet. Genomics. 2010; 284: 95-103Crossref PubMed Scopus (158) Google Scholar). Recent findings of individual miRNAs expressed during spermatogenesis in a developmental stage-specific manner suggest the participation of miRNAs in male germ cell development through their contribution to cell type-specific profiles of protein expression during spermatogenesis (16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar, 17Ro S. Park C. Sanders K.M. McCarrey J.R. Yan W. Dev. Biol. 2007; 311: 592-602Crossref PubMed Scopus (202) Google Scholar).To elucidate regulatory actions of GRTH in miRNA processing during germ cell development, we first compared the differential expression profiles of miRNA in purified round spermatids from wild type and GRTH null mice. A panel of miRNAs and some of their corresponding pri-miRNAs was significantly increased in the GRTH-null mice. Among the various up-regulated miRNAs, the testis-specific miR-469 with cell expression similar to GRTH was selected to pursue further studies (16Yu Z. Raabe T. Hecht N.B. Biol. Reprod. 2005; 73: 427-433Crossref PubMed Scopus (193) Google Scholar). miR-469 repressed the translation of TP2 and Prm2 through binding to the coding region of TP2 and Prm2 mRNAs. This is consistent with the preservation of TP2 and Prm2 mRNA with the failure of their protein expression found in the GRTH null mice (7Sheng Y. Tsai-Morris C.H. Gutti R. Maeda Y. Dufau M.L. J. Biol. Chem. 2006; 281: 35048-35056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Also, GRTH appears to negatively regulate Drosha/DGCR8 gene expression to control microRNA maturation. This study has provided insights into a novel molecular control mechanism of GRTH through the microRNA pathway in the regulation of spermatogenesis.
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