Kinesin KIF18A is a novel PUM-regulated target promoting mitotic progression and survival of a human male germ cell line.

Regulation of proliferation, apoptosis and cell cycle is crucial for the physiology of germ cells. Their malfunction contributes to infertility and germ cell tumours. The kinesin KIF18A is an important regulator of those processes in animal germ cells. Post-transcriptional regulation of KIF18A has not been extensively explored. Owing to the presence of PUM-binding elements (PBEs), KIF18A mRNA is a potential target of PUM proteins, where PUM refers to Pumilio proteins, RNA-binding proteins that act in post-transcriptional gene regulation. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assays, by applying an appropriate luciferase construct encoding wild-type KIF18A 3'-UTR, upon PUM overexpression or knockdown in TCam-2 cells, representing human male germ cells. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function, an MTS proliferation assay, and apoptosis and cell cycle analysis using flow cytometry, was performed upon KIF18A mRNA siRNA knockdown. KIF18A significantly influences proliferation, apoptosis and the cell cycle, with its effects being opposite to PUM effects. Repression by PUM proteins might represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells.

Emerging Roles of NANOS RNA-Binding Proteins in Cancer.

In recent years, growing evidence demonstrates that mammalian Nanos RNA-binding proteins (Nanos1, Nanos2, and Nanos3), known for their indispensable roles in germline development, are overexpressed in a variety of cancers. This overexpression contributes to various oncogenic properties including cancer growth, invasiveness, and metastasis. Here, we highlight recent findings regarding the role of mammalian Nanos RNA-binding proteins and the mechanisms of their overexpression in cancer. In addition, we present expression profiles of human NANOS genes and their oncogenic transcriptional regulators obtained from publicly available cancer and normal tissue RNA-Seq datasets. Altogether, we emphasize the functional significance of NANOS proteins across human cancers as well as highlight the missing links to understanding the full scope of their role in carcinogenesis.

Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model.

Nanos RNA-binding proteins are critical factors of germline development throughout the animal kingdom and their dysfunction causes infertility. During evolution, mammalian Nanos paralogues adopted divergent roles in germ cell biology. However, the molecular basis behind this divergence, such as their target mRNAs, remains poorly understood. Our RNA-sequencing analysis in a human primordial germ cell model-TCam-2 cell line revealed distinct pools of genes involved in the cell cycle process downregulated upon NANOS1 and NANOS3 overexpression. We show that NANOS1 and NANOS3 proteins influence different stages of the cell cycle. Namely, NANOS1 is involved in the G1/S and NANOS3 in the G2/M phase transition. Many of their cell cycle targets are known infertility and cancer-germ cell genes. Moreover, NANOS3 in complex with RNA-binding protein PUM1 causes 3'UTR-mediated repression of FOXM1 mRNA encoding a transcription factor crucial for G2/M phase transition. Interestingly, while NANOS3 and PUM1 act as post-transcriptional repressors of FOXM1, FOXM1 potentially acts as a transcriptional activator of NANOS3, PUM1, and itself. Finally, by utilizing publicly available RNA-sequencing datasets, we show that the balance between FOXM1-NANOS3 and FOXM1-PUM1 expression levels is disrupted in testis cancer, suggesting a potential role in this disease.

PUM1 and PUM2 exhibit different modes of regulation for SIAH1 that involve cooperativity with NANOS paralogues.

Pumilio (PUM) proteins are RNA-binding proteins that posttranscriptionally regulate gene expression in many organisms. Their PUF domain recognizes specific PUM-binding elements (PBE) in the 3' untranslated region of target mRNAs while engaging protein cofactors such as NANOS that repress the expression of target mRNAs through the recruitment of effector complexes. Although the general process whereby PUM recognizes individual mRNAs has been studied extensively, the particulars of the mechanism underlying PUM-NANOS cooperation in mRNA regulation and the functional overlap among PUM and NANOS paralogues in mammals have not been elucidated. Here, using the novel PUM1 and PUM2 mRNA target SIAH1 as a model, we show mechanistic differences between PUM1 and PUM2 and between NANOS1, 2, and 3 paralogues in the regulation of SIAH1. Specifically, unlike PUM2, PUM1 exhibited PBE-independent repression of SIAH1 3'UTR-dependent luciferase expression. Concordantly, the PUF domains of PUM1 and PUM2 showed different EMSA complex formation patterns with SIAH1 3'UTRs. Importantly, we show direct binding of NANOS3, but not NANOS2, to SIAH1 3'UTR, which did not require PBEs or the PUF domain. To the best of our knowledge, this is the first report, showing that an NANOS protein directly binds RNA. Finally, using NANOS1 and NANOS3 constructs carrying mutations identified in infertile patients, we show that these mutations disrupt repression of the SIAH1-luciferase reporter and that the central region in NANOS1 appears to contribute to the regulation of SIAH1. Our findings highlight the mechanistic versatility of the PUM/NANOS machinery in mammalian posttranscriptional regulation.

The FKBP4 Gene, Encoding a Regulator of the Androgen Receptor Signaling Pathway, Is a Novel Candidate Gene for Androgen Insensitivity Syndrome.

Androgen insensitivity syndrome (AIS), manifesting incomplete virilization in 46,XY individuals, is caused mostly by androgen receptor (AR) gene mutations. Therefore, a search for AR mutations is a routine approach in AIS diagnosis. However, some AIS patients lack AR mutations, which complicates the diagnosis. Here, we describe a patient suffering from partial androgen insensitivity syndrome (PAIS) and lacking AR mutations. The whole exome sequencing of the patient and his family members identified a heterozygous FKBP4 gene mutation, c.956T>C (p.Leu319Pro), inherited from the mother. The gene encodes FKBP prolyl isomerase 4, a positive regulator of the AR signaling pathway. This is the first report describing a FKBP4 gene mutation in association with a human disorder of sexual development (DSD). Importantly, the dysfunction of a homologous gene was previously reported in mice, resulting in a phenotype corresponding to PAIS. Moreover, the Leu319Pro amino acid substitution occurred in a highly conserved position of the FKBP4 region, responsible for interaction with other proteins that are crucial for the AR functional heterocomplex formation and therefore the substitution is predicted to cause the disease. We proposed the FKBP4 gene as a candidate AIS gene and suggest screening that gene for the molecular diagnosis of AIS patients lacking AR gene mutations.

Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line.

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes.

Mutations of NANOS1, a human homologue of the Drosophila morphogen, are associated with a lack of germ cells in testes or severe oligo-astheno-teratozoospermia.

BACKGROUND: The Nanos gene is a key translational regulator of specific mRNAs involved in Drosophila germ cell development. Disruption of mammalian homologues, Nanos2 or Nanos3, causes male infertility in mice. In humans, however, no evidence of NANOS2 or NANOS3 mutations causing male infertility has been reported. Although Nanos1 seems dispensable for mouse reproduction, we sought to analyse for the first time its homologue in infertile men. METHODS: A group of 195 patients manifesting non-obstructive azoospermia or oligozoospermia were tested for mutations of the NANOS1 gene, using single-strand conformation polymorphism and DNA sequencing. RESULTS: Three types of NANOS1 gene mutations were identified in five patients and were absent in 800 chromosomes of fertile men. Pedigree analysis indicated a dominant inheritance pattern with penetration limited to males. Two mutations caused deletions of single amino acids, p.Pro77_Ser78delinsPro and p.Ala173del, each of them identified in two unrelated patients. Both types of deletions were located in the NANOS1 N-terminus (responsible for protein interactions) and were associated with a lack of germ cells in testes. Interestingly, the Pro77_Ser78delinsPro mutation altered interaction of NANOS1 with a microRNA biogenesis factor, GEMIN3. The third identified mutation, p.[(Arg246His; Arg276Tyr)], found in the C-terminal RNA-binding domain, was present in a single oligo-astheno-teratozoospermic man. We bioinformatically demonstrated that the p.Arg246His substitution causes a decrease in the positive charge of this domain, potentially altering RNA-binding. CONCLUSIONS: This is the first report describing the association of NANOS1 gene mutations with human infertility. Two different infertility phenotypes may reflect distinct functions of N-terminal versus C-terminal regions of NANOS1.

Mutations in STARD8 (DLC3) May Cause 46,XY Gonadal Dysgenesis.

INTRODUCTION: 46,XY gonadal dysgenesis is a condition that is characterised by undeveloped testes in individuals with a male karyotype. Mutations in many genes that underlie this condition have been identified; however, there are still a considerable number of patients with an unknown genetic background. Recently, a mutation in the STARD8 X-linked gene in two sisters with 46,XY gonadal dysgenesis has been reported. It was localised within the START domain, whose homologue in Drosophila is responsible for maintaining testes integrity during their development. METHODS: We analysed the potential pathogenicity of another STARD8 mutation, p.R887C, that was identified in a patient with 46,XY asymmetric gonadal dysgenesis. For this purpose, molecular dynamics simulations were performed. RESULTS: These simulations revealed the full rearrangement of the helix containing the p.R887C substitution upstream from the START domain, which may cause STARD8 protein dysfunction and contribute to 46,XY gonadal dysgenesis. A comparison of the phenotypes of the three described 46,XY gonadal dysgenesis patients that harbour STARD8 mutations indicated that alterations of this gene can result in a partial or complete gonadal dysgenesis phenotype. CONCLUSION: Based on these and previous results, it is reasonable to include STARD8 in gene panels for 46,XY gonadal dysgenesis.

A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9.

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for -KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no -KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/-KTS ratio.

Characterization of RNP Networks of PUM1 and PUM2 Post-Transcriptional Regulators in TCam-2 Cells, a Human Male Germ Cell Model.

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins (RBPs) with wide-ranging roles. They are involved in germ cell development, which has functional implications in development and fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity. To address that problem, first we used RIP-Seq and RNA-Seq approaches, and identified mRNA pools regulated by PUM1 and PUM2 proteins in the TCam-2 cell line, a human male germ cell model. Second, applying global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-interacting putative protein cofactors, most of them involved in RNA processing. Third, combinatorial analysis of RIP and RNA-Seq, mass spectrometry, and RNA motif enrichment analysis revealed that PUM1 and PUM2 form partially varied RNP-regulatory networks (RNA regulons), which indicate different roles in human reproduction and testicular tumorigenesis. Altogether, this work proposes that protein paralogues with very similar and evolutionary highly conserved functional domains may play divergent roles in the cell by combining with different sets of protein cofactors. Our findings highlight the versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

The gene encoding the ketogenic enzyme HMGCS2 displays a unique expression during gonad development in mice.

Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.

A Case of Two Sisters Suffering from 46,XY Gonadal Dysgenesis and Carrying a Mutation of a Novel Candidate Sex-Determining Gene STARD8 on the X Chromosome.

Identification of novel genes involved in sexual development is crucial for understanding disorders of sex development (DSD). Here, we propose a member of the START domain family, the X chromosome STARD8, as a DSD candidate gene. We have identified a missense mutation of this gene in 2 sisters with 46,XY gonadal dysgenesis, inherited from their heterozygous mother. Gonadal tissue of one of the sisters contained Leydig cells overloaded with cholesterol droplets, i.e., structures previously identified in 46,XY DSD patients carrying mutations in the STAR gene encoding another START domain family member, which is crucial for steroidogenesis. Based on the phenotypes of our patients, we propose a dual role of STARD8 in sexual development, namely in testes determination and testosterone synthesis. However, further studies are needed to confirm the involvement of STARD8 in sexual development.

A Case of Wiedemann-Steiner Syndrome Associated with a 46,XY Disorder of Sexual Development and Gonadal Dysgenesis.

We report the case of a female patient suffering from a 46,XY disorder of sexual development (DSD) with complete gonadal dysgenesis and Wiedemann-Steiner Syndrome (WDSTS). The coexistence of these 2 conditions has not yet been reported. Using whole exome sequencing and comparative genome hybridization array, we identified a de novo MLL/KMT2A gene nonsense mutation which explains the WDSTS phenotype. In addition, we discovered novel genetic variants, which could explain the testicular dysgenesis observed in the patient, a maternally inherited 167-kb duplication of DAAM2 and MOCS1 genes and a de novo LRRC33/NRROS gene mutation. These genes, some of which are expressed during mouse gonadal development, could be considered as potentially new candidate genes for DSD.