An atlas of chromatoid body components

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells

GermOnline 4.0 is a genomics gateway for germline development, meiosis and the mitotic cell cycle

GermOnline 4.0 is a cross-species database portal focusing on high-throughput expression data relevant for germline development, the meiotic cell cycle and mitosis in healthy versus malignant cells. It is thus a source of information for life scientists as well as clinicians who are interested in gene expression and regulatory networks. The GermOnline gateway provides unlimited access to information produced with high-density oligonucleotide microarrays (3′-UTR GeneChips), genome-wide protein–DNA binding assays and protein–protein interaction studies in the context of Ensembl genome annotation. Samples used to produce high-throughput expression data and to carry out genome-wide in vivo DNA binding assays are annotated via the MIAME-compliant Multiomics Information Management and Annotation System (MIMAS 3.0). Furthermore, the Saccharomyces Genomics Viewer (SGV) was developed and integrated into the gateway. SGV is a visualization tool that outputs genome annotation and DNA-strand specific expression data produced with high-density oligonucleotide tiling microarrays (Sc_tlg GeneChips) which cover the complete budding yeast genome on both DNA strands. It facilitates the interpretation of expression levels and transcript structures determined for various cell types cultured under different growth and differentiation conditions

SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3 ' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.Peer reviewe

The Annotation, Mapping, Expression and Network (AMEN) suite of tools for molecular systems biology

<p>Abstract</p> <p>Background</p> <p>High-throughput genome biological experiments yield large and multifaceted datasets that require flexible and user-friendly analysis tools to facilitate their interpretation by life scientists. Many solutions currently exist, but they are often limited to specific steps in the complex process of data management and analysis and some require extensive informatics skills to be installed and run efficiently.</p> <p>Results</p> <p>We developed the Annotation, Mapping, Expression and Network (AMEN) software as a stand-alone, unified suite of tools that enables biological and medical researchers with basic bioinformatics training to manage and explore genome annotation, chromosomal mapping, protein-protein interaction, expression profiling and proteomics data. The current version provides modules for (i) uploading and pre-processing data from microarray expression profiling experiments, (ii) detecting groups of significantly co-expressed genes, and (iii) searching for enrichment of functional annotations within those groups. Moreover, the user interface is designed to simultaneously visualize several types of data such as protein-protein interaction networks in conjunction with expression profiles and cellular co-localization patterns. We have successfully applied the program to interpret expression profiling data from budding yeast, rodents and human.</p> <p>Conclusion</p> <p>AMEN is an innovative solution for molecular systems biological data analysis freely available under the GNU license. The program is available via a website at the Sourceforge portal which includes a user guide with concrete examples, links to external databases and helpful comments to implement additional functionalities. We emphasize that AMEN will continue to be developed and maintained by our laboratory because it has proven to be extremely useful for our genome biological research program.</p

℮-conome: an automated tissue counting platform of cone photoreceptors for rodent models of retinitis pigmentosa

<p>Abstract</p> <p>Background</p> <p>Retinitis pigmentosa is characterized by the sequential loss of rod and cone photoreceptors. The preservation of cones would prevent blindness due to their essential role in human vision. Rod-derived Cone Viability Factor is a thioredoxin-like protein that is secreted by rods and is involved in cone survival. To validate the activity of Rod-derived Cone Viability Factors (RdCVFs) as therapeutic agents for treating retinitis Pigmentosa, we have developed e-conome, an automated cell counting platform for retinal flat mounts of rodent models of cone degeneration. This automated quantification method allows for faster data analysis thereby accelerating translational research.</p> <p>Methods</p> <p>An inverted fluorescent microscope, motorized and coupled to a CCD camera records images of cones labeled with fluorescent peanut agglutinin lectin on flat-mounted retinas. In an average of 300 fields per retina, nine Z-planes at magnification X40 are acquired after two-stage autofocus individually for each field. The projection of the stack of 9 images is subject to a threshold, filtered to exclude aberrant images based on preset variables. The cones are identified by treating the resulting image using 13 variables empirically determined. The cone density is calculated over the 300 fields.</p> <p>Results</p> <p>The method was validated by comparison to the conventional stereological counting. The decrease in cone density in <it>rd1 </it>mouse was found to be equivalent to the decrease determined by stereological counting. We also studied the spatiotemporal pattern of the degeneration of cones in the <it>rd1 </it>mouse and show that while the reduction in cone density starts in the central part of the retina, cone degeneration progresses at the same speed over the whole retinal surface. We finally show that for mice with an inactivation of the Nucleoredoxin-like genes <it>Nxnl1 </it>or <it>Nxnl2 </it>encoding RdCVFs, the loss of cones is more pronounced in the ventral retina.</p> <p>Conclusion</p> <p>The automated platform ℮-conome used here for retinal disease is a tool that can broadly accelerate translational research for neurodegenerative diseases.</p

Temporally Regulated Traffic of HuR and Its Associated ARE-Containing mRNAs from the Chromatoid Body to Polysomes during Mouse Spermatogenesis

International audienceBACKGROUND: In mammals, a temporal disconnection between mRNA transcription and protein synthesis occurs during late steps of germ cell differentiation, in contrast to most somatic tissues where transcription and translation are closely linked. Indeed, during late stages of spermatogenesis, protein synthesis relies on the appropriate storage of translationally inactive mRNAs in transcriptionally silent spermatids. The factors and cellular compartments regulating mRNA storage and the timing of their translation are still poorly understood. The chromatoid body (CB), that shares components with the P. bodies found in somatic cells, has recently been proposed to be a site of mRNA processing. Here, we describe a new component of the CB, the RNA binding protein HuR, known in somatic cells to control the stability/translation of AU-rich containing mRNAs (ARE-mRNAs). METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of cell imagery and sucrose gradient fractionation, we show that HuR localization is highly dynamic during spermatid differentiation. First, in early round spermatids, HuR colocalizes with the Mouse Vasa Homolog, MVH, a marker of the CB. As spermatids differentiate, HuR exits the CB and concomitantly associates with polysomes. Using computational analyses, we identified two testis ARE-containing mRNAs, Brd2 and GCNF that are bound by HuR and MVH. We show that these target ARE-mRNAs follow HuR trafficking, accumulating successively in the CB, where they are translationally silent, and in polysomes during spermatid differentiation. CONCLUSIONS/SIGNIFICANCE: Our results reveal a temporal regulation of HuR trafficking together with its target mRNAs from the CB to polysomes as spermatids differentiate. They strongly suggest that through the transport of ARE-mRNAs from the CB to polysomes, HuR controls the appropriate timing of ARE-mRNA translation. HuR might represent a major post-transcriptional regulator, by promoting mRNA storage and then translation, during male germ cell differentiation

The Nucleoside Diphosphate Kinase Gene Nme3 Acts as Quantitative Trait Locus Promoting Non-Mendelian Inheritance

The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (>95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas +-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOKTcr, a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa

SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3 ' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility

3640 Unique EST Clusters from the Medaka Testis and Their Potential Use for Identifying Conserved Testicular Gene Expression in Fish and Mammals

BACKGROUND: The fish medaka is the first vertebrate capable of full spermatogenesis in vitro from self-renewing spermatogonial stem cells to motile test-tube sperm. Precise staging and molecular dissection of this process has been hampered by the lack of suitable molecular markers. METHODOLOGY AND PRINCIPAL FINDINGS: We have generated a normalized medaka testis cDNA library and obtained 7040 high quality sequences representing 3641 unique gene clusters. Among these, 1197 unique clusters are homologous to known genes, and 2444 appear to be novel genes. Ontology analysis shows that the 1197 gene products are implicated in diverse molecular and cellular processes. These genes include markers for all major types of testicular somatic and germ cells. Furthermore, markers were identified for major spermatogenic stages ranging from spermatogonial stem cell self-renewal to meiosis entry, progression and completion. Intriguingly, the medaka testis expresses at least 13 homologs of the 33 mouse X-chromosomal genes that are enriched in the testis. More importantly, we show that key components of several signaling pathways known to be important for testicular function in mammals are well represented in the medaka testicular EST collection. CONCLUSIONS/SIGNIFICANCE: Medaka exhibits a considerable similarity in testicular gene expression to mammals. The medaka testicular EST collection we obtained has wide range coverage and will not only consolidate our knowledge on the comparative analysis of known genes' functions in the testis but also provide a rich resource to dissect molecular events and mechanism of spermatogenesis in vivo and in vitro in medaka as an excellent vertebrate model

A comprehensive functional analysis of tissue specificity of human gene expression

<p>Abstract</p> <p>Background</p> <p>In recent years, the maturation of microarray technology has allowed the genome-wide analysis of gene expression patterns to identify tissue-specific and ubiquitously expressed ('housekeeping') genes. We have performed a functional and topological analysis of housekeeping and tissue-specific networks to identify universally necessary biological processes, and those unique to or characteristic of particular tissues.</p> <p>Results</p> <p>We measured whole genome expression in 31 human tissues, identifying 2374 housekeeping genes expressed in all tissues, and genes uniquely expressed in each tissue. Comprehensive functional analysis showed that the housekeeping set is substantially larger than previously thought, and is enriched with vital processes such as oxidative phosphorylation, ubiquitin-dependent proteolysis, translation and energy metabolism. Network topology of the housekeeping network was characterized by higher connectivity and shorter paths between the proteins than the global network. Ontology enrichment scoring and network topology of tissue-specific genes were consistent with each tissue's function and expression patterns clustered together in accordance with tissue origin. Tissue-specific genes were twice as likely as housekeeping genes to be drug targets, allowing the identification of tissue 'signature networks' that will facilitate the discovery of new therapeutic targets and biomarkers of tissue-targeted diseases.</p> <p>Conclusion</p> <p>A comprehensive functional analysis of housekeeping and tissue-specific genes showed that the biological function of housekeeping and tissue-specific genes was consistent with tissue origin. Network analysis revealed that tissue-specific networks have distinct network properties related to each tissue's function. Tissue 'signature networks' promise to be a rich source of targets and biomarkers for disease treatment and diagnosis.</p