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.

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.

NANOS1 and PUMILIO2 bind microRNA biogenesis factor GEMIN3, within chromatoid body in human germ cells.

Nanos and pumilio bind each other to regulate translation of specific mRNAs in germ cells of model organisms, such as D. melanogaster or C. elegans. Recently described human homologues NANOS1 and PUMILIO2 form a complex similar to their ancestors. This study was aimed to identify the proteins interacting with NANOS1-PUMILIO2 complex in the human spermatogenic cells. Here, using the yeast two-hybrid system we found that NANOS1 and PUMILIO2 proteins interact with RNA DEAD-box helicase GEMIN3, a microRNA biogenesis factor. Moreover, GEMIN3 coimmunoprecipitates with NANOS1 and PUMILIO2 in transfected mammalian cells. By double immunofluorescence staining, we observed that complexes built of NANOS1, PUMILIO2 and GEMIN3 are located within cytoplasmic region of germ cells. These proteins condense to form a compact aggregate in the round spermatids of the human and mouse germ cells. This aggregate was reminiscent of the chromatoid body (CB), a perinuclear structure present in the mammalian male germ line. This structure is considered evolutionary remnant of germ plasm, a hallmark structure of germ cells in lower metazoan. Using a CB marker VASA protein, we demonstrated that CBs are present in the human round spermatids, as they are in the mouse. Moreover, NANOS1, PUMILIO2 and GEMIN3 colocalize with VASA protein. We demonstrated for the first time that a mammalian Nanos-Pumilio complex functions within CB, a center of RNA storing and processing, involving microRNAs. NANOS1-PUMILIO2 complex, together with GEMIN3 and small noncoding RNAs, possibly regulate mRNA translation within CB of the human germ cells.

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.

SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

SPIN1 is necessary for normal meiotic progression in mammals. It is overexpressed in human ovarian cancers and some cancer cell lines. Here, we examined the functional significance and regulation of SPIN1 and SPIN3 in the TCam-2 human seminoma cell line. We found that while SPIN1 overexpression reduced apoptosis in these cells, SPIN3 overexpression induced it. Similarly, SPIN1 upregulated and SPIN3 downregulated CYCD1, which is a downstream target of the PI3K/AKT pathway and contributes to apoptosis resistance in cancer cell lines. It appears that SPIN1 is pro-oncogenic and SPIN3 acts as a tumor suppressor in TCam-2 cells. To our knowledge, this is the first report of SPIN3 tumor suppressor activity. However, both SPIN1 and SPIN3 stimulated cell cycle progression. In addition, using luciferase reporters carrying SPIN1 or SPIN3 mRNA 3'UTRs, we found that PUM1 and PUM2 targeted and repressed SPINs. We also found that PUM1 itself strongly stimulated apoptosis and moderately slowed cell cycle progression in TCam-2 cells, suggesting that PUM1, like SPIN3, is a tumor suppressor. Our findings suggest that acting, at least in part, through SPIN1 and SPIN3, PUM proteins contribute to a mechanism promoting normal human male germ cell apoptotic status and thus preventing cancer.

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.

Human fertility protein PUMILIO2 interacts in vitro with testis mRNA encoding Cdc42 effector 3 (CEP3).

PUMILIO protein regulates translation of specific mRNAs in morphogenesis and in development of the germ-line of model organisms such as flies and worms. Given that a human homologue (PUMILIO2) was recently identified in the germ-line stem cells, the question was raised whether it regulates translation of fertility mRNAs similarly to Drosophila Pumilio. Here, we describe a candidate mRNA encoding Cdc42 effector protein 3 (CEP3), however, a function for this protein in reproduction has previously not been reported. We detected three CEP3 transcripts in the testis tissue including one which was highly expressed and testis specific by northern blotting. We found that CEP mRNA contains GUUGU (A) and AUUGUA (B) motifs (ABB) within the 3' untranslated region (3'UTR), which are also present in mRNA targets of Pumilio in Drosophila. Interaction of PUMILIO2 with the fragment of CEP3 transcript containing the ABB array was tested by mobility shift assay and we found that PUMILIO2 binds the 3' untranslated region of the CEP3 mRNA. These results support the hypothesis that CEP3 mRNA may be a target of PUMILIO2 protein in the human male gonad and be under translational control mediated by specific nucleotide motifs within the 3'UTR.

Candidate mRNAs interacting with fertility protein PUMILIO2 in the human germ line.

Pumilio protein regulates translation of specific mRNAs in morphogenesis and germ-line development of the flies by binding nucleotide motifs GUUGU (A) and AUUGUA (B) in 3'untranslated regions. A human homologue, PUMILIO2 has been recently identified in the germ-line stem cells and the question was raised whether it regulates translation. We designed software to screen the GeneBank for A and B motifs and found that they are not uncommon in the human genome. Moreover, some of the genes containing motifs A and B are germ cell specific, but some others are expressed in a number of other tissues. This may indicate that PUMILIO mediated translational regulation is universally used in developmental processes of the human body.

Conservation of a Pumilio-Nanos complex from Drosophila germ plasm to human germ cells.

Germ cells are the cells which ultimately give rise to mature sperm and eggs. In model organisms such as flies and worms, several genes that are required for formation and maintenance of germ cells have been identified and their interactions are rapidly being delineated. By contrast, little is known of the genes required for development of human germ cells and it is not clear whether findings from model organisms will translate into knowledge of human germ cell development, especially given observations that reproductive pathways may evolve more rapidly than somatic pathways. The Pumilio and Nanos genes have been especially well-characterized in model organisms and encode proteins that interact and are required for development of germ stem cells in one or both sexes. Here we report the first characterization of a mammalian Nanos homolog, human NANOS1 ( NOS1). We show that human NOS1 protein interacts with the human PUMILIO-2 (PUM2) protein via highly conserved domains to form a stable complex. We also show that in men, the NOS1 and PUM2 proteins are particularly abundant in germline stem cells. These observations mirror those in distant species and document for the first time a conserved protein-protein interaction in germ cells from flies to humans. These results suggest the possibility that the interaction of PUM2 and NOS1 may play a conserved role in germ cell development and maintenance in humans as in model organisms.