Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

The Definition of Open Reading Frame Revisited.

The term open reading frame (ORF) is of central importance to gene finding. Surprisingly, at least three definitions are in use. We discuss several molecular biological and bioinformatics aspects, and we recommend using the definition in which an ORF is bounded by stop codons.

Long-lived rodents reveal signatures of positive selection in genes associated with lifespan.

The genetics of lifespan determination is poorly understood. Most research has been done on short-lived animals and it is unclear if these insights can be transferred to long-lived mammals like humans. Some African mole-rats (Bathyergidae) have life expectancies that are multiple times higher than similar sized and phylogenetically closely related rodents. To gain new insights into genetic mechanisms determining mammalian lifespans, we obtained genomic and transcriptomic data from 17 rodent species and scanned eleven evolutionary branches associated with the evolution of enhanced longevity for positively selected genes (PSGs). Indicating relevance for aging, the set of 250 identified PSGs showed in liver of long-lived naked mole-rats and short-lived rats an expression pattern that fits the antagonistic pleiotropy theory of aging. Moreover, we found the PSGs to be enriched for genes known to be related to aging. Among these enrichments were "cellular respiration" and "metal ion homeostasis", as well as functional terms associated with processes regulated by the mTOR pathway: translation, autophagy and inflammation. Remarkably, among PSGs are RHEB, a regulator of mTOR, and IGF1, both central components of aging-relevant pathways, as well as genes yet unknown to be aging-associated but representing convincing functional candidates, e.g. RHEBL1, AMHR2, PSMG1 and AGER. Exemplary protein homology modeling suggests functional consequences for amino acid changes under positive selection. Therefore, we conclude that our results provide a meaningful resource for follow-up studies to mechanistically link identified genes and amino acids under positive selection to aging and lifespan determination.

The pseudogenes of barley.

Pseudogenes have a reputation of being 'evolutionary relics' or 'junk DNA'. While they are well characterized in mammals, studies in more complex plant genomes have so far been hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high-quality genome reference assembly available for a Triticeae crop, we conducted a whole-genome assessment of pseudogenes on the barley genome. We identified, characterized and classified 89 440 gene fragments and pseudogenes scattered along the chromosomes, with occasional hotspots and higher densities at the chromosome ends. Full-length pseudogenes (11 015) have preferentially retained their exon-intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints at founding mechanisms. While parent genes related to the defense-response were found to be under-represented in cultivated barley, we detected several defense-related pseudogenes in wild barley accessions. The percentage of transcriptionally active pseudogenes is 7.2%, and these may potentially adopt new regulatory roles.The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non-random duplication and pseudogenization preferences and improve our understanding of the dynamics of gene birth and death in large plant genomes and the mechanisms that lead to evolutionary innovations.

Correction to: A miRNA catalogue and ncRNA annotation of the short-living fish Nothobranchius furzeri.

Following the publication of this article [1], the authors reported that the images of Figs. 1, 2 and 3 were published in the incorrect order, whereby they mismatch with their captions.

The Wilms tumor protein Wt1 contributes to female fertility by regulating oviductal proteostasis.

Although the zinc finger transcription factor Wt1 has been linked to female fertility, its precise role in this process has not yet been understood. We have sequenced the WT1 exons in a panel of patients with idiopathic infertility and have identified a missense mutation in WT1 in one patient out of eight. This mutation leads to an amino acid change within the zinc finger domain and results in reduced DNA binding. We utilized Wt1+/- mice as a model to mechanistically pinpoint the consequences of reduced Wt1 levels for female fertility. Our results indicate that subfertility in Wt1+/- female mice is a maternal effect caused by the Wt1-dependent de-regulation of Prss29, encoding a serine protease. Notably, blocking Prss29 activity was sufficient to rescue subfertility in Wt1+/- mice indicating Prss29 as a critical factor in female fertility. Molecularly, Wt1 represses expression of Prss29. De-repression and precocious expression of Prss29 in the oviduct of Wt1+/- mice interferes with pre-implantation development. Our study reveals a novel role for Wt1 in early mammalian development and identifies proteases as critical mediators of the maternal-embryonic interaction. Our data also suggest that the role of Wt1 in regulating fertility is conserved in mammals.

Nothobranchius furzeri: A Model for Aging Research and More.

The short-lived killifish Nothobranchius furzeri inhabits ephemeral ponds in southeastern Africa and is characterized by rapid growth and early sexual maturation. With respect to the molecular, cellular, and integrative traits of aging, N. furzeri shows significant similarities to mammals, including humans. Recently, reference sequences for the N. furzeri genome have been published. Also, methods for transgenesis and genomic engineering have been established. In this review we discuss why the killifish is a valuable model for aging research and what we have learned from the genome sequence. The respective insights are not limited to the biology of aging but are also relevant for developmental biology and the evolution of sex determination.

Outgroups and Positive Selection: The Nothobranchius furzeri Case.

Applications of positive selection analysis increase with the number of species for which genome/transcriptome sequences become available. Using the recently sequenced turquoise killifish (Nothobranchius furzeri) genome as an example, we compare two different approaches based on different outgroup selection. The combination of these two methods allows the origin of positively selected sites in aging-related genes of the N. furzeri genome to be determined.

Smg6/Est1 licenses embryonic stem cell differentiation via nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional mechanism that targets aberrant transcripts and regulates the cellular RNA reservoir. Genetic modulation in vertebrates suggests that NMD is critical for cellular and tissue homeostasis, although the underlying mechanism remains elusive. Here, we generate knockout mice lacking Smg6/Est1, a key nuclease in NMD and a telomerase cofactor. While the complete loss of Smg6 causes mouse lethality at the blastocyst stage, inducible deletion of Smg6 is compatible with embryonic stem cell (ESC) proliferation despite the absence of telomere maintenance and functional NMD. Differentiation of Smg6-deficient ESCs is blocked due to sustained expression of pluripotency genes, normally repressed by NMD, and forced down-regulation of one such target, c-Myc, relieves the differentiation block. Smg6-null embryonic fibroblasts are viable as well, but are refractory to cellular reprograming into induced pluripotent stem cells (iPSCs). Finally, depletion of all major NMD factors compromises ESC differentiation, thus identifying NMD as a licensing factor for the switch of cell identity in the process of stem cell differentiation and somatic cell reprograming.

PosiGene: automated and easy-to-use pipeline for genome-wide detection of positively selected genes.

Many comparative genomics studies aim to find the genetic basis of species-specific phenotypic traits. A prevailing strategy is to search genome-wide for genes that evolved under positive selection based on the non-synonymous to synonymous substitution ratio. However, incongruent results largely due to high false positive rates indicate the need for standardization of quality criteria and software tools. Main challenges are the ortholog and isoform assignment, the high sensitivity of the statistical models to alignment errors and the imperative to parallelize large parts of the software. We developed the software tool PosiGene that (i) detects positively selected genes (PSGs) on genome-scale, (ii) allows analysis of specific evolutionary branches, (iii) can be used in arbitrary species contexts and (iv) offers visualization of the results for further manual validation and biological interpretation. We exemplify PosiGene's performance using simulated and real data. In the simulated data approach, we determined a false positive rate <1%. With real data, we found that 68.4% of the PSGs detected by PosiGene, were shared by at least one previous study that used the same set of species. PosiGene is a user-friendly, reliable tool for reproducible genome-wide identification of PSGs and freely available at https://github.com/gengit/PosiGene.

Naked mole-rat transcriptome signatures of socially suppressed sexual maturation and links of reproduction to aging.

BACKGROUND: Naked mole-rats (NMRs) are eusocially organized in colonies. Although breeders carry the additional metabolic load of reproduction, they are extremely long-lived and remain fertile throughout their lifespan. This phenomenon contrasts the disposable soma theory of aging stating that organisms can invest their resources either in somatic maintenance, enabling a longer lifespan, or in reproduction, at the cost of longevity. Here, we present a comparative transcriptome analysis of breeders vs. non-breeders of the eusocial, long-lived NMR vs. the polygynous and shorter-lived guinea pig (GP). RESULTS: Comparative transcriptome analysis of tissue samples from ten organs showed, in contrast to GPs, low levels of differentiation between sexes in adult NMR non-breeders. After transition into breeders, NMR transcriptomes are markedly sex-specific, show pronounced feedback signaling via gonadal steroids, and have similarities to reproductive phenotypes in African cichlid fish, which also exhibit social status changes between dominant and subordinate phenotypes. Further, NMRs show functional enrichment of status-related expression differences associated with aging. Lipid metabolism and oxidative phosphorylation-molecular networks known to be linked to aging-were identified among most affected gene sets. Remarkably and in contrast to GPs, transcriptome patterns associated with longevity are reinforced in NMR breeders. CONCLUSION: Our results provide comprehensive and unbiased molecular insights into interspecies differences between NMRs and GPs, both in sexual maturation and in the impact of reproduction on longevity. We present molecular evidence that sexual maturation in NMRs is socially suppressed. In agreement with evolutionary theories of aging in eusocial organisms, we have identified transcriptome patterns in NMR breeders that-in contrast to the disposable soma theory of aging-may slow down aging rates and potentially contribute to their exceptional long life- and healthspan.

Species comparison of liver proteomes reveals links to naked mole-rat longevity and human aging.

BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.

3D Network exploration and visualisation for lifespan data.

BACKGROUND: The Ageing Factor Database AgeFactDB contains a large number of lifespan observations for ageing-related factors like genes, chemical compounds, and other factors such as dietary restriction in different organisms. These data provide quantitative information on the effect of ageing factors from genetic interventions or manipulations of lifespan. Analysis strategies beyond common static database queries are highly desirable for the inspection of complex relationships between AgeFactDB data sets. 3D visualisation can be extremely valuable for advanced data exploration. RESULTS: Different types of networks and visualisation strategies are proposed, ranging from basic networks of individual ageing factors for a single species to complex multi-species networks. The augmentation of lifespan observation networks by annotation nodes, like gene ontology terms, is shown to facilitate and speed up data analysis. We developed a new Javascript 3D network viewer JANet that provides the proposed visualisation strategies and has a customised interface for AgeFactDB data. It enables the analysis of gene lists in combination with AgeFactDB data and the interactive visualisation of the results. CONCLUSION: Interactive 3D network visualisation allows to supplement complex database queries by a visually guided exploration process. The JANet interface allows gaining deeper insights into lifespan data patterns not accessible by common database queries alone. These concepts can be utilised in many other research fields.

A physical, genetic and functional sequence assembly of the barley genome.

Barley (Hordeum vulgare L.) is among the world's earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 'high-confidence' genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

Tissue-, sex-, and age-specific DNA methylation of rat glucocorticoid receptor gene promoter and insulin-like growth factor 2 imprinting control region.

Tissue-, sex-, and age-specific epigenetic modifications such as DNA methylation are largely unknown. Changes in DNA methylation of the glucocorticoid receptor gene (NR3C1) and imprinting control region (ICR) of IGF2 and H19 genes during the lifespan are particularly interesting since these genes are susceptible to epigenetic modifications by prenatal stress or malnutrition. They are important regulators of development and aging. Methylation changes of NR3C1 affect glucocorticoid receptor expression, which is associated with stress sensitivity and stress-related diseases predominantly occurring during aging. Methylation changes of IGF2/H19 affect growth trajectory and nutrient use with risk of metabolic syndrome. Using a locus-specific approach, we characterized DNA methylation patterns of different Nr3c1 promoters and Igf2/H19 ICR in seven tissues of rats at 3, 9, and 24 mo of age. We found a complex pattern of locus-, tissue-, sex-, and age-specific DNA methylation. Tissue-specific methylation was most prominent at the shores of the Nr3c1 CpG island (CGI). Sex-specific differences in methylation peaked at 9 mo. During aging, Nr3c1 predominantly displayed hypomethylation mainly in females and at shores, whereas hypermethylation occurred within the CGI. Igf2/H19 ICR exhibited age-related hypomethylation occurring mainly in males. Methylation patterns of Nr3c1 in the skin correlated with those in the cortex, hippocampus, and hypothalamus. Skin may serve as proxy for methylation changes in central parts of the hypothalamic-pituitary-adrenal axis and hence for vulnerability to stress- and age-associated diseases. Thus, we provide in-depth insight into the complex DNA methylation changes of rat Nr3c1 and Igf2/H19 during aging that are tissue and sex specific.

A miRNA catalogue and ncRNA annotation of the short-living fish Nothobranchius furzeri.

BACKGROUND: The short-lived fish Nothobranchius furzeri is the shortest-lived vertebrate that can be cultured in captivity and was recently established as a model organism for aging research. Small non-coding RNAs, especially miRNAs, are implicated in age dependent control of gene expression. RESULTS: Here, we present a comprehensive catalogue of miRNAs and several other non-coding RNA classes (ncRNAs) for Nothobranchius furzeri. Analyzing multiple small RNA-Seq libraries, we show most of these identified miRNAs are expressed in at least one of seven Nothobranchius species. Additionally, duplication and clustering of N. furzeri miRNAs was analyzed and compared to the four fish species Danio rerio, Oryzias latipes, Gasterosteus aculeatus and Takifugu rubripes. A peculiar characteristic of N. furzeri, as compared to other teleosts, was a duplication of the miR-29 cluster. CONCLUSION: The completeness of the catalogue we provide is comparable to that of the zebrafish. This catalogue represents a basis to investigate the role of miRNAs in aging and development in this species.

FRAMA: from RNA-seq data to annotated mRNA assemblies.

BACKGROUND: Advances in second-generation sequencing of RNA made a near-complete characterization of transcriptomes affordable. However, the reconstruction of full-length mRNAs via de novo RNA-seq assembly is still difficult due to the complexity of eukaryote transcriptomes with highly similar paralogs and multiple alternative splice variants. Here, we present FRAMA, a genome-independent annotation tool for de novo mRNA assemblies that addresses several post-assembly tasks, such as reduction of contig redundancy, ortholog assignment, correction of misassembled transcripts, scaffolding of fragmented transcripts and coding sequence identification. RESULTS: We applied FRAMA to assemble and annotate the transcriptome of the naked mole-rat and assess the quality of the obtained compilation of transcripts with the aid of publicy available naked mole-rat gene annotations. Based on a de novo transcriptome assembly (Trinity), FRAMA annotated 21,984 naked mole-rat mRNAs (12,100 full-length CDSs), corresponding to 16,887 genes. The scaffolding of 3488 genes increased the median sequence information 1.27-fold. In total, FRAMA detected and corrected 4774 misassembled genes, which were predominantly caused by fusion of genes. A comparison with three different sources of naked mole-rat transcripts reveals that FRAMA's gene models are better supported by RNA-seq data than any other transcript set. Further, our results demonstrate the competitiveness of FRAMA to state of the art genome-based transcript reconstruction approaches. CONCLUSION: FRAMA realizes the de novo construction of a low-redundant transcript catalog for eukaryotes, including the extension and refinement of transcripts. Thereby, results delivered by FRAMA provide the basis for comprehensive downstream analyses like gene expression studies or comparative transcriptomics. FRAMA is available at https://github.com/gengit/FRAMA .

Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses.

BACKGROUND: The Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts. RESULTS: The genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type. CONCLUSION: Both cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.

BACKGROUND: Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. RESULTS: Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. CONCLUSION: We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.