Metazoan Genomes Research Articles

Fertility of hybrids of dromedary and Bactrian camels: A possible role of conserved architecture of zinc finger domain of recombination regulator PRDM9

Recombination regulator, PRDM9, has been regarded as the most rapidly evolving gene in the genomes of many metazoans, in addition to being acknowledged as the sole speciation gene in vertebrates. It has become the focus of many scientific investigations because of exceptional numerical and sequence variability in its zinc finger (ZF) domain within and across species that contributes to reproductive isolation between species. This study is the maiden attempt to explore the architecture of PRDM9 ZF domain in two Camelid species (Camelus dromedarius and Camelus bactrianus). Sequence analysis revealed highly conserved domain architecture with presence of 3 and 4 ZFs in dromedary and Bactrian camels, respectively. Typical evolutionary features of PRDM9 ZF domain i.e. concerted evolution and positive selection were invariably absent in both the one-humped dromedary and the two-humped Bactrian camels. Fertility of hybrids of dromedary and Bactrian camels, despite being taxonomically distinct species can be attributed to the lack of sequence variability in PRDM9 in these species. Phylogenetic analysis underpinned clear demarcation of camels from other livestock species. The results of the present study defy what has been learnt so far about PRDM9 and add to the enigma surrounding the most intriguing gene in the genome.

The compact genome of Caenorhabditis niphades n. sp., isolated from a wood-boring weevil, Niphades variegatus

BackgroundThe first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation.ResultsIn the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica.ConclusionsThe compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

Tree2GD: a phylogenomic method to detect large-scale gene duplication events.

Whole-genome duplication events have long been discovered throughout the evolution of eukaryotes, contributing to genome complexity and biodiversity and leaving traces in the descending organisms. Therefore, an accurate and rapid phylogenomic method is needed to identify the retained duplicated genes on various lineages across the target taxonomy. Here, we present Tree2GD, an integrated method to identify large-scale gene duplication events by automatically perform multiple procedures, including sequence alignment, recognition of homolog, gene tree/species tree reconciliation, Ks distribution of gene duplicates and synteny analyses. Application of Tree2GD on 2 datasets, 12 metazoan genomes and 68 angiosperms, successfully identifies all reported whole-genome duplication events exhibited by these species, showing effectiveness and efficiency of Tree2GD on phylogenomic analyses of large-scale gene duplications. Tree2GD is written in Python and C++ and is available at https://github.com/Dee-chen/Tree2gd. Supplementary data are available at Bioinformatics online.

Comparative Genome and Evolution Analyses of an Endangered Stony Coral Species Dendrophyllia cribrosa Near Dokdo Islands in the East Sea.

Stony corals often harbor intracellular photosynthetic dinoflagellate algae that receive dissolved inorganic nutrients. However, Dendrophyllia cribrosa is a nonsymbiotic stony coral distributed in the western Pacific. We assembled a chromosome-level D. cribrosa genome using PacBio and Hi-C technologies. The final assembly was 625 Mb, distributed on 14 chromosomes, and contained 30,493 protein-coding genes. The Benchmarking Universal Single-Copy Orthologs analysis revealed a percentage of 96.8 of the metazoan genome. A comparative phylogenetic analysis revealed that D. cribrosa, which lacks symbionts, evolved to acquire cellular energy by expanding genes related to acyl-CoA metabolism and carbohydrate transporters. This species also has expanded immune-related genes involved in the receptor protein tyrosine kinase signaling pathway. In addition, we observed a specific expansion of calcification genes, such as coral acid-rich proteins and carbonic anhydrase, in D. cribrosa. This high-quality reference genome and comparative analysis provides insights into the ecology and evolution of nonsymbiotic stony corals.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Use of 6 Nucleotide Length Words to Study the Complexity of Gene Sequences from Different Organisms.

In this paper, we attempted to find a relation between bacteria living conditions and their genome algorithmic complexity. We developed a probabilistic mathematical method for the evaluation of k-words (6 bases length) occurrence irregularity in bacterial gene coding sequences. For this, the coding sequences from different bacterial genomes were analyzed and as an index of k-words occurrence irregularity, we used , which has a distribution similar to normal. The research results for bacterial genomes show that they can be divided into two uneven groups. First, the smaller one has W in the interval from 170 to 475, while for the second it is from 475 to 875. Plants, metazoan and virus genomes also have W in the same interval as the first bacterial group. We suggested that second bacterial group coding sequences are much less susceptible to evolutionary changes than the first group ones. It is also discussed to use the W index as a biological stress value.

The Expression and Function of Tubulin Isotypes in Caenorhabditis elegans

Microtubules, made from the polymerization of the highly conserved α/β-tubulin heterodimers, serve as important components of the cytoskeleton in all eukaryotic cells. The existence of multiple tubulin isotypes in metazoan genomes and a dazzling variety of tubulin posttranslational modifications (PTMs) prompted the “tubulin code” hypothesis, which proposed that microtubule structure and functions are determined by the tubulin composition and PTMs. Evidence for the tubulin code has emerged from studies in several organisms with the characterization of specific tubulins for their expression and functions. The studies of tubulin PTMs are accelerated by the discovery of the enzymes that add or remove the PTMs. In tubulin research, the use of simple organisms, such as Caenorhabditis elegans, has been instrumental for understanding the expression and functional specialization of tubulin isotypes and the effects of their PTMs. In this review, we summarize the current understanding of the expression patterns and cellular functions of the nine α-tubulin and six β-tubulin isotypes. Expression studies are greatly facilitated by the CRISPR/Cas9-mediated endogenous GFP knock-in reporters and the organism-wide single cell transcriptomic studies. Meanwhile, functional studies benefit from the ease of genetic manipulation and precise gene replacement in C. elegans. These studies identified both ubiquitously expressed tubulin isotypes and tissue-specific isotypes. The isotypes showed functional redundancy, as well as functional specificity, which is likely caused by the subtle differences in their amino acid sequences. Many of these differences concentrate at the C-terminal tails that are subjected to several PTMs. Indeed, tubulin PTM, such as polyglutamylation, is shown to modulate microtubule organization and properties in both ciliated and non-ciliated neurons. Overall, studies from C. elegans support the distinct expression and function patterns of tubulin isotypes and the importance of their PTMs and offer the promise of cracking the tubulin code at the whole-genome and the whole-organism level.

A glimpse on metazoan ZNFX1 helicases, ancient players of antiviral innate immunity

The human zinc finger NFX1-type containing 1 (ZNFX1) is an interferon-stimulated protein associated to the outer mitochondrial membrane, able to bind dsRNAs and interact with MAVS proteins, promoting type I IFN response in the early stage of viral infection. An N-terminal Armadillo (ARM)-type fold and a large helicase core (P-loop) and zinc fingers confer RNA-binding and ATPase activities to ZNFX1. We studied the phylogenetic distribution of metazoan ZNFX1s, ZNFX1 gene expression trends and genomic and protein signatures during viral infection of invertebrates. Based on 221 ZNFX1 sequences, we obtained a polyphyletic tree with a taxonomy-consistent branching at the phylum-level only. In metazoan genomes, ZNFX1 genes were found either in single copy, with up to some tens of exons in vertebrates, or in multiple copies, with one or a few exons and one of them sometimes encompassing most of the coding sequence, in invertebrates like sponges, sea urchins and mollusks. Structural analyses of selected ZNFX1 proteins showed high conservation of the helicase region (P-loop), an overall conserved region and domain architecture, an ARM-fold mostly traceable, and the presence of intrinsically disordered regions of varying length and position. The remarkable over-expression of ZNFX1 in bivalve and gastropod mollusks infected with dsDNA viruses underscores the antiviral role of ZNFX1, whereas nothing similar was found in virus-infected nematodes and corals. Whether the functional diversification reported in the C. elegans ZNFX1 occurs in other metazoan proteins remains to be established.

Evidence for exon shuffling is sensitive to model choice.

The exon shuffling theory posits that intronic recombination creates new domain combinations, facilitating the evolution of novel protein function. This theory predicts that introns will be preferentially situated near domain boundaries. Many studies have sought evidence for exon shuffling by testing the correspondence between introns and domain boundaries against chance intron positioning. Here, we present an empirical investigation of how the choice of null model influences significance. Although genome-wide studies have used a uniform null model, exclusively, more realistic null models have been proposed for single gene studies. We extended these models for genome-wide analyses and applied them to 21 metazoan and fungal genomes. Our results show that compared with the other two models, the uniform model does not recapitulate genuine exon lengths, dramatically underestimates the probability of chance agreement, and overestimates the significance of intron-domain correspondence by as much as 100 orders of magnitude. Model choice had much greater impact on the assessment of exon shuffling in fungal genomes than in metazoa, leading to different evolutionary conclusions in seven of the 16 fungal genomes tested. Genome-wide studies that use this overly permissive null model may exaggerate the importance of exon shuffling as a general mechanism of multidomain evolution.

Ensembl Genomes 2022: an expanding genome resource for non-vertebrates.

Ensembl Genomes (https://www.ensemblgenomes.org) provides access to non-vertebrate genomes and analysis complementing vertebrate resources developed by the Ensembl project (https://www.ensembl.org). The two resources collectively present genome annotation through a consistent set of interfaces spanning the tree of life presenting genome sequence, annotation, variation, transcriptomic data and comparative analysis. Here, we present our largest increase in plant, metazoan and fungal genomes since the project's inception creating one of the world's most comprehensive genomic resources and describe our efforts to reduce genome redundancy in our Bacteria portal. We detail our new efforts in gene annotation, our emerging support for pangenome analysis, our efforts to accelerate data dissemination through the Ensembl Rapid Release resource and our new AlphaFold visualization. Finally, we present details of our future plans including updates on our integration with Ensembl, and how we plan to improve our support for the microbial research community. Software and data are made available without restriction via our website, online tools platform and programmatic interfaces (available under an Apache 2.0 license). Data updates are synchronised with Ensembl's release cycle.

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations.

Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism’s fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains.

Pegasus, a small extracellular peptide enhancing short-range diffusion of Wingless

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions.

Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence invivo.

SUMMARYThe nuclear lamina is typically associated with transcriptional silencing, and peripheral relocation of genes highly correlates with repression. However, the DNA sequences and proteins regulating gene-lamina interactions are largely unknown. Exploiting the developmentally timed hunchback gene movement to the lamina in Drosophila neuroblasts, we identified a 250 bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, we found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, our results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing.

The enigmatic Placozoa part 1: Exploring evolutionary controversies and poor ecological knowledge.

The placozoan Trichoplax adhaerens is a tiny hairy plate and more simply organized than any other living metazoan. After its original description by F.E. Schulze in 1883, it attracted attention as a potential model for the ancestral state of metazoan organization, the "Urmetazoon". Trichoplax lacks any kind of symmetry, organs, nerve cells, muscle cells, basal lamina, and extracellular matrix. Furthermore, the placozoan genome is the smallest (not secondarily reduced) genome of all metazoan genomes. It harbors a remarkably rich diversity of genes and has been considered the best living surrogate for a metazoan ancestor genome. The phylum Placozoa presently harbors three formally described species, while several dozen "cryptic" species are yet awaiting their description. The phylogenetic position of placozoans has recently become a contested arena for modern phylogenetic analyses and view-driven claims. Trichoplax offers unique prospects for understanding the minimal requirements of metazoan animal organization and their corresponding malfunctions.

The regulation of ferroptosis by MESH1 through the activation of the integrative stress response

All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation “alarmone” (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response. However, the function of MESH1 remains unknown until our recent discovery of MESH1 as the first cytosolic NADPH phosphatase that regulates ferroptosis. To further understand whether MESH1 knockdown triggers a similar transcriptional response in mammalian cells, here, we employed RNA-Seq to analyze the transcriptome response to MESH1 knockdown in human cancer cells. We find that MESH1 knockdown induced different genes involving endoplasmic reticulum (ER) stress, especially ATF3, one of the ATF4-regulated genes in the integrative stress responses (ISR). Furthermore, MESH1 knockdown increased ATF4 protein, eIF2a phosphorylation, and induction of ATF3, XBPs, and CHOP mRNA. ATF4 induction contributes to ~30% of the transcriptome induced by MESH1 knockdown. Concurrent ATF4 knockdown re-sensitizes MESH1-depleted RCC4 cells to ferroptosis, suggesting its role in the ferroptosis protection mediated by MESH1 knockdown. ATF3 induction is abolished by the concurrent knockdown of NADK, implicating a role of NADPH accumulation in the integrative stress response. Collectively, these results suggest that MESH1 depletion triggers ER stress and ISR as a part of its overall transcriptome changes to enable stress survival of cancer cells. Therefore, the phenotypic similarity of stress tolerance caused by MESH1 removal and NADPH accumulation is in part achieved by ISR to regulate ferroptosis.

Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture.

Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabeling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here, we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of long-terminal repeat retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.

Complete mitochondrial genomes of the two Glycyphagoidea mites Lepidoglyphus destructor and Gohieria fusca (Acari: Sarcoptiformes, Glycyphagoidea): Revelation of a novel feature of the largest non-coding region

Mitochondrial genomes (mitogenomes) of metazoans typically contain 37 genes, comprising 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. To date, complete mitogenome sequences of 15 species of Astigmatina have been elucidated; and they contain variations in several features, such as gene order, unconventional tRNA secondary structures, and the number and internal structures of control regions. Here, we sequenced the mitogenomes of Lepidoglyphus destructor and Gohieria fusca, thus representing the first reported complete mitogenome sequence of mite species from the superfamily Glycyphagoidea. In total, 37 mitochondrial genes were identified in both species. The same gene arrangement pattern was found in both species, and this pattern was consistent with that in the possible common ancestor of astigmatid mites. Most tRNAs were highly truncated in both species, which is typical of acariform mites. The largest non-coding region in L. destructor and G. fusca can be divided into two domains based on AT content and stem-loop structures. Domain I has a stem-loop structure; domain II is more AT-rich than domain I and includes several conserved sequences that are also observed in Acaridae and Pyroglyphidae. The largest non-coding region of Acalvolia sp. (Astigmatina, Hemisarcoptoidea) also exhibits similar features. The phylogenetic relationships found in our study are consistent with those identified by previous phylogenetic studies on astigmatid mites. Within Astigmatina, Glycyphagoidea was recovered as a monophyletic group.

DNA molecular combing-based replication fork directionality profiling.

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

Accurate and sensitive detection of microbial eukaryotes from whole metagenome shotgun sequencing

BackgroundMicrobial eukaryotes are found alongside bacteria and archaea in natural microbial systems, including host-associated microbiomes. While microbial eukaryotes are critical to these communities, they are challenging to study with shotgun sequencing techniques and are therefore often excluded.ResultsHere, we present EukDetect, a bioinformatics method to identify eukaryotes in shotgun metagenomic sequencing data. Our approach uses a database of 521,824 universal marker genes from 241 conserved gene families, which we curated from 3713 fungal, protist, non-vertebrate metazoan, and non-streptophyte archaeplastida genomes and transcriptomes. EukDetect has a broad taxonomic coverage of microbial eukaryotes, performs well on low-abundance and closely related species, and is resilient against bacterial contamination in eukaryotic genomes. Using EukDetect, we describe the spatial distribution of eukaryotes along the human gastrointestinal tract, showing that fungi and protists are present in the lumen and mucosa throughout the large intestine. We discover that there is a succession of eukaryotes that colonize the human gut during the first years of life, mirroring patterns of developmental succession observed in gut bacteria. By comparing DNA and RNA sequencing of paired samples from human stool, we find that many eukaryotes continue active transcription after passage through the gut, though some do not, suggesting they are dormant or nonviable. We analyze metagenomic data from the Baltic Sea and find that eukaryotes differ across locations and salinity gradients. Finally, we observe eukaryotes in Arabidopsis leaf samples, many of which are not identifiable from public protein databases.ConclusionsEukDetect provides an automated and reliable way to characterize eukaryotes in shotgun sequencing datasets from diverse microbiomes. We demonstrate that it enables discoveries that would be missed or clouded by false positives with standard shotgun sequence analysis. EukDetect will greatly advance our understanding of how microbial eukaryotes contribute to microbiomes.EDH-t7L4v5WEFMvCw3ubMSVideo abstract

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.