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

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 GRTHnull 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.Mammalian 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 (1, 2). 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 2 / DDX25), a testis-specific member of the DEAD (Asp-Glu-GlyAsp)-box family present in Leydig and germ cells (meiotic spermatocytes and round spermatids) is regulated developmentally by androgen at the transcriptional level (3-6). 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 stor...

MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

“…Microarray analysis was used to determine and compare the miRNA expression patterns in infertile sperm and normal sperm. Based on previous investigations [20][21][22], we chose miR-122 as the object of study, and attempted to identify its role in the maturation and development of spermatozoa. We found that human iPS cells miR-122 with miR-122 could be induced to differentiate into human spermatozoa-like cells more efficiently than mutant miR-122-transfected cells.…”

“…Yu et al first reported that miR-122 negatively regulates Tnp2 mRNA by endonucleolytic cleavage, due to specific base pair interactions of miR-122 with its complementary binding sequence in the 3¢-untranslated region (3¢-UTR) of Tnp2 mRNA [21]. Meanwhile, Dai et al also revealed that expression of testisspecific miR-469 bound to the coding region and repressed the translation of Tnp2 and Prm2 mRNA, to regulate the development and maturation of sperm cells [20].…”

“…MicroRNAs (miRNAs), a class of *22-nucleotide (nt) noncoding RNAs, participate in diverse biological functions by promoting the degradation or inhibiting the translation of their target mRNAs [19][20][21][22][23][24][25][26][27]. Recent findings that individual miRNAs are expressed during spermatogenesis in a developmental stage-specific manner suggest that miRNAs participate in male germ cell development, by contributing to cell type-specific protein expression profiles during spermatogenesis [20]. Yu et al first reported that miR-122 negatively regulates Tnp2 mRNA by endonucleolytic cleavage, due to specific base pair interactions of miR-122 with its complementary binding sequence in the 3¢-untranslated region (3¢-UTR) of Tnp2 mRNA [21].…”

Role of microRNAs in spermatogenesis

“…Было по-казано, что мишенью для микроРНК-469 являются мРНК транзиторного белка ТР2 и мРНК протамина Prm2, в результате чего подавляется экспрессия белка TP2 и Prm2 в пахитенных сперматоцитах и ранних спер-матидах. Другая микроРНК, контролирующая экспрес-сию гена Tр2, -микроРНК-122а, эта микро РНК ак-тивирует деградацию мРНК Тр2 [82][83][84]. Микро РНК-18, принадлежащая онкогенному микроРНК-кластеру -Oncomir-1 -регулирует трансляцию транскрипцион-ного фактора HSF2 во время сперматогенеза [85,86].…”

Review: Role of microRNAs during spermatogenesis in mammals

“…Interestingly, in contrast of miR-469 the miR-122a also can bind with Tp2 on 3'-UTR which induced mRNA cleavage and lead to the abnormal spermatozoa in experimental mice [78]. Furthermore, Fatime et al, (2014) has investigated that the following miRNAs (including miR-34a, -181b, -469 and -122a) altering their expression level by exposure of tertiarybutyl hydroperoxide (TBHP) and suggested that are these miRNAs were involved in cellular functions including cell cycle and apoptosis [79], moreover consistency in the expression level about said miRNAs has been found in previous studies [16,79]. Recently, Liu et al, (2015) reported 173 DE miRNAs profile between spermatocytes and spermatids in human azoospermic patients, among those miR-34b-5p and miR-34c-5p were significantly upregulated in spermatocytes as compared with round spermatids and it was suggested that these miRNAs are critical at meiosis phase of spermatocytes [17].…”