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Background Vertebrates and insects diverged approximately 700 million years ago, and yet they retain a large core of conserved genes from their last common ancestor. These ancient genes present strong evolutionary constraints, which limit their overall sequence and expression divergence. However, these constraints can greatly vary across ancestral gene families and, in at least some cases, sequence and expression changes can have functional consequences. Importantly, overall patterns of sequence and expression divergence and their potential functional outcomes have never been explored in a genome-wide manner across large animal evolutionary distances. Results We focus on approximately 7000 highly conserved genes shared between vertebrates and insects, and we investigate global patterns of molecular diversification driven by changes in sequence and gene expression. We identify molecular features generally linked to higher or lower diversification rates, together with gene groups with similar diversification profiles in both clades. Moreover, we discover that specific sets of genes underwent differential diversification during vertebrate and insect evolution, potentially contributing to the emergence of unique phenotypes in each clade. Conclusions We generate a comprehensive dataset of measures of sequence and expression divergence across vertebrates and insects, which reveals a continuous spectrum of evolutionary constraints among highly conserved genes. These constraints are normally consistent between these two clades and are associated with specific molecular features, but in some cases we also identify instances of lineage-specific diversification likely linked to functional evolution.

Background Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. Results We generate a massive resource for Arabidopsis thaliana , PastDB , comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana , being also largely involved in modulating gene expression through NMD and uORF inclusion. Conclusions Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana .

Circadian and circannual cycles trigger physiological changes whose reflection on human transcriptomes remains largely uncharted. We used the time and season of death of 932 individuals from GTEx to jointly investigate transcriptomic changes associated with those cycles across multiple tissues. Overall, most variation across tissues during day-night and among seasons was unique to each cycle. Although all tissues remodeled their transcriptomes, brain and gonadal tissues exhibited the highest seasonality, whereas those in the thoracic cavity showed stronger day-night regulation. Core clock genes displayed marked day-night differences across multiple tissues, which were largely conserved in baboon and mouse, but adapted to their nocturnal or diurnal habits. Seasonal variation of expression affected multiple pathways, and it was enriched among genes associated with the immune response, consistent with the seasonality of viral infections. Furthermore, they unveiled cytoarchitectural changes in brain regions. Altogether, our results provide the first combined atlas of how transcriptomes from human tissues adapt to major cycling environmental conditions. This atlas may have multiple applications; for example, drug targets with day-night or seasonal variation in gene expression may benefit from temporally adjusted doses.

Two waves of DNA methylation reprogramming occur during mammalian embryogenesis; during preimplantation development and during primordial germ cell (PGC) formation. However, it is currently unclear how evolutionarily conserved these processes are. Here we characterise the DNA methylomes of zebrafish PGCs at four developmental stages and identify retention of paternal epigenetic memory, in stark contrast to the findings in mammals. Gene expression profiling of zebrafish PGCs at the same developmental stages revealed that the embryonic germline is defined by a small number of markers that display strong developmental stage-specificity and that are independent of DNA methylation-mediated regulation. We identified promoters that are specifically targeted by DNA methylation in somatic and germline tissues during vertebrate embryogenesis and that are frequently misregulated in human cancers. Together, these detailed methylome and transcriptome maps of the zebrafish germline provide insight into vertebrate DNA methylation reprogramming and enhance our understanding of the relationships between germline fate acquisition and oncogenesis. Germ cells are the means of transferring genetic information to the next generation. Here the authors characterise the DNA methylomes of zebrafish primordial germ cells and find that, unlike mammals, the zebrafish germ cells do not undergo genome-wide DNA demethylation but rather retain paternal DNA methylation patterns

The evolution of winged insects revolutionized terrestrial ecosystems and led to the largest animal radiation on Earth. However, we still have an incomplete picture of the genomic changes that underlay this diversification. Mayflies, as one of the sister groups of all other winged insects, are key to understanding this radiation. Here, we describe the genome of the mayfly Cloeon dipterum and its gene expression throughout its aquatic and aerial life cycle and specific organs. We discover an expansion of odorant-binding-protein genes, some expressed specifically in breathing gills of aquatic nymphs, suggesting a novel sensory role for this organ. In contrast, flying adults use an enlarged opsin set in a sexually dimorphic manner, with some expressed only in males. Finally, we identify a set of wing-associated genes deeply conserved in the pterygote insects and find transcriptomic similarities between gills and wings, suggesting a common genetic program. Globally, this comprehensive genomic and transcriptomic study uncovers the genetic basis of key evolutionary adaptations in mayflies and winged insects. Genomic studies of paleopteran insects, such as mayflies, are needed to reconstruct early insect evolution. Here, Almudi and colleagues present the genome of the mayfly Cloeon dipterum and use transcriptomics to characterize its adaptations to distinct habitats and the origin of insect wings.

Background Alternative splicing, particularly through intron retention and exon skipping, is a major layer of pre-translational regulation in eukaryotes. While intron retention is believed to be the most prevalent mode across non-animal eukaryotes, animals have unusually high rates of exon skipping. However, when and how this high prevalence of exon skipping evolved is unknown. Since exon skipping can greatly expand proteomes, answering these questions sheds light on the evolution of higher organismal complexity in metazoans. Results We used RNA-seq data to quantify exon skipping and intron retention frequencies across 65 eukaryotic species, with particular focus on early branching animals and unicellular holozoans. We found that only bilaterians have significantly increased their exon skipping frequencies compared to all other eukaryotic groups. Unlike in other eukaryotes, however, exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving sequences, suggesting that exon skipping involvement in proteome expansion predated the increase in frequency. We also identified architectural features consistently associated with higher exon skipping rates within all studied eukaryotic genomes. Remarkably, these architectures became more prevalent during animal evolution, indicating co-evolution between genome architectures and exon skipping frequencies. Conclusion We suggest that the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes.

The evolution of metazoans from their unicellular ancestors was one of the most important events in the history of life. However, the cellular and genetic changes that ultimately led to the evolution of multicellularity are not known. In this study, we describe an aggregative multicellular stage in the protist Capsaspora owczarzaki, a close unicellular relative of metazoans. Remarkably, transition to the aggregative stage is associated with significant upregulation of orthologs of genes known to establish multicellularity and tissue architecture in metazoans. We further observe transitions in regulated alternative splicing during the C. owczarzaki life cycle, including the deployment of an exon network associated with signaling, a feature of splicing regulation so far only observed in metazoans. Our results reveal the existence of a highly regulated aggregative stage in C. owczarzaki and further suggest that features of aggregative behavior in an ancestral protist may had been co-opted to develop some multicellular properties currently seen in metazoans. When living things made from many cells evolved from single-celled ancestors, it was a breakthrough in the history of life—and one that has occurred more than once. In fact, multicellular life has evolved independently at least 25 times, in groups as diverse as animals, fungi, plants, slime molds and seaweeds. There are broadly two ways to become multicellular. The most complex multicellular species, such as animals, will replicate a single cell, over and over, without separating the resultant cells. However, in species that are only occasionally multicellular, free-living cells tend instead to join together in one mass of many cells. Evolution is constrained by its raw materials; so looking at the living relatives of a given species, or group, can lead to a better understanding of its evolution because its relatives contain clues about its ancestors. To gain insights into how animal multicellular life might have began; Sebé-Pedrós et al. studied the life cycle of the amoeboid organism Capsaspora owczarzaki. Found within the bodies of freshwater snails, this single-celled amoeba is a close relative of multicellular animals and could resemble one of their earliest ancestors. At certain stages of the life cycle Sebé-Pedrós et al. noticed that individual amoebae gathered together to form a multicellular mass—something that had not been seen before in such a close relative of the animals. Moreover, the genes that ‘switched on’ when the amoebae began to aggregate are also found in animals; where, together with other genes, they control development and the formation of tissues. Sebé-Pedrós et al. suggest that the first multicellular animals could have recycled the genes that control the aggregation of single-celled species: in other words, genes that once controlled the changes that happen at different times in a life cycle, now control the changes that develop between different tissues at the same time. Sebé-Pedrós et al. also observed that alternative splicing—a process that allows different proteins to be made from a single gene—occurs via two different mechanisms during the life cycle of Capsaspora. Most of the time Capsaspora employs a form of alternative splicing that is often seen in plants and fungi, and only rarely in animals; for the rest of the time it uses a form of alternative splicing similar to that used by animal cells. The evolution of complex alternative splicing mechanisms is a hallmark feature of multicellular animals. The exploitation of two major forms of alternative splicing in Capsaspora could thus reflect an important transition during evolution that resulted in an increased diversity of proteins and in more complex gene regulation. Such a transition may ultimately have paved the way for the increased specialization of cell types seen in animals. This glimpse into the possible transitions in gene regulation that contributed to the birth of multicellular animals indicates that the single-celled ancestor of the animals was likely more complex than previously thought. Future analyses of the animals’ close relatives may further improve our understanding of how single-celled organisms became multicellular animals.

Several splicing-modulating compounds, including Sudemycins and Spliceostatin A, display anti-tumor properties. Combining transcriptome, bioinformatic and mutagenesis analyses, we delineate sequence determinants of the differential sensitivity of 3′ splice sites to these drugs. Sequences 5′ from the branch point (BP) region strongly influence drug sensitivity, with additional functional BPs reducing, and BP-like sequences allowing, drug responses. Drug-induced retained introns are typically shorter, displaying higher GC content and weaker polypyrimidine-tracts and BPs. Drug-induced exon skipping preferentially affects shorter alternatively spliced regions with weaker BPs. Remarkably, structurally similar drugs display both common and differential effects on splicing regulation, SSA generally displaying stronger effects on intron retention, and Sudemycins more acute effects on exon skipping. Collectively, our results illustrate how splicing modulation is exquisitely sensitive to the sequence context of 3′ splice sites and to small structural differences between drugs. Several families of natural compounds target core components of the pre-mRNA splicing machinery and display anti-tumor activity. Here the authors show that particular sequence features can be linked to drug response, and that drugs with very similar chemical structures display substantially different effects on splicing regulation.

Single-cell sequencing technologies are revolutionizing biology, but they are limited by the need to dissociate live samples. Here, we present ACME (ACetic-MEthanol), a dissociation approach for single-cell transcriptomics that simultaneously fixes cells. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, and are sortable and permeable. As a proof of principle, we provide single-cell transcriptomic data of different species, using both droplet-based and combinatorial barcoding single-cell methods. ACME uses affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.

How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ∼350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species.