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Key Points The recent advent of high-throughput sequencing-based approaches enables detailed qualitative and quantitative comparisons of transcriptomes and gene regulatory mechanisms between distant species. Initial transcriptome comparisons based on RNA sequencing have revealed different rates of expression evolution for different types of transcripts, mammalian lineages and organs due to global differences in selective pressures. The differentiation of mammalian sex chromosomes from ordinary ancestral autosomes entailed substantial and chromosome-wide remodelling of gene contents and expression patterns owing to the emerging sex-related selective forces. The search for adaptive expression changes is challenging because of various confounding factors. Nevertheless, various phenotypically relevant expression changes have been identified, which warrant further experimental validation and characterization. Although regulatory mechanisms underlying gene expression are now fairly well understood overall, only a few studies have attempted to jointly analyse transcriptome evolution and the underlying regulatory changes. These initial studies suggest that adaptive gene expression evolution may be driven by both cis - and trans -regulatory changes. One of the most challenging but also most important future research directions is the detailed experimental characterization of detected transcriptome changes, which will ultimately reveal their actual biological and evolutionary relevance. This Review provides insights obtained from comparative transcriptomic studies of mammalian species. The dynamics of gene expression evolution in coding and non-coding genes, as well as the regulatory basis of transcriptome evolution and future research avenues, are discussed. Gene expression changes may underlie much of phenotypic evolution. The development of high-throughput RNA sequencing protocols has opened the door to unprecedented large-scale and cross-species transcriptome comparisons by allowing accurate and sensitive assessments of transcript sequences and expression levels. Here, we review the initial wave of the new generation of comparative transcriptomic studies in mammals and vertebrate outgroup species in the context of earlier work. Together with various large-scale genomic and epigenomic data, these studies have unveiled commonalities and differences in the dynamics of gene expression evolution for various types of coding and non-coding genes across mammalian lineages, organs, developmental stages, chromosomes and sexes. They have also provided intriguing new clues to the regulatory basis and phenotypic implications of evolutionary gene expression changes.

Alternative splicing (AS) is pervasive in mammalian genomes, yet cross-species comparisons have been largely restricted to adult tissues and the functionality of most AS events remains unclear. We assessed AS patterns across pre- and postnatal development of seven organs in six mammals and a bird. Our analyses revealed that developmentally dynamic AS events, which are especially prevalent in the brain, are substantially more conserved than nondynamic ones. Cassette exons with increasing inclusion frequencies during development show the strongest signals of conserved and regulated AS. Newly emerged cassette exons are typically incorporated late in testis development, but those retained during evolution are predominantly brain specific. Our work suggests that an intricate interplay of programs controlling gene expression levels and AS is fundamental to organ development, especially for the brain and heart. In these regulatory networks, AS affords substantial functional diversification of genes through the generation of tissue- and time-specific isoforms from broadly expressed genes. Analysis of RNA-seq datasets from seven organs across seven species generates an alternative splicing (AS) atlas and shows that AS events provide functional gene diversification through generation of tissue- and time-specific transcript isoforms.

Key Points The enzymatic machinery encoded by certain retrotransposons enables genes to duplicate via an RNA intermediate, a mechanism termed retroposition or retroduplication. In mammals, retroposition has produced thousands of gene copies, termed retrocopies. Retrocopies are expected to lack promoter sequences and were long regarded as pseudogenes with no functional relevance. However, cases of functional retrocopies, termed retrogenes, have accumulated in the literature, implying that retrocopies can be transcribed. Recent large-scale studies indicate that transcribed retrocopies are widespread. Retrocopies can become transcribed in various ways. For example, they can use promoters of other genes or retrotransposable elements in their vicinity, but they can (unexpectedly) also inherit promoters from their parental source genes. Retrocopies and retrogenes are frequently functionally transcribed in the testis, which is probably due to the permissive transcriptional state of chromatin during and after meiosis. Several 'out-of-the-X' autosomal retrogenes have been shown to functionally substitute their X-linked parental genes during and after meiotic sex-chromosome inactivation. Phylogenetic dating of out-of-the-X retrogenes in mammals has led to the reassessment of the age of our sex chromosomes. Detailed functional studies of young retrogenes have provided novel insights pertaining to the origin of new genes. For example, analyses of recent primate genes revealed that new gene functions can arise through changes in the localization of encoded proteins in the cell during evolution, whereas studies in Drosophila melanogaster uncovered the first example of a new gene with a behavioral phenotype. Studies of the process of retroposition have not only shed light on the origin of new genes, but have also provided other general insights pertaining to the evolution of mammalian genomes. For example, retrocopies have served as unique 'genomic archives' of mammalian transcriptomes, revealing extinct transcripts and gene expression activity during evolution. Gene copies originating from segmental duplication and retroposition have distinct features (such as the presence or absence of inherited regulatory sequences and introns) that profoundly influence their evolutionary fate. Studying RNA-based gene duplication is therefore a useful alternative to further enhance our understanding of the emergence of new genes and their functions. Gene duplication can occur via insertion of reverse transcribed mRNAs into the genome. Although originally thought to be non-functional, recent studies have uncovered how these retrocopies can acquire novel functions, and how patterns of retroposition can give unexpected insights into genome evolution. Gene copies that stem from the mRNAs of parental source genes have long been viewed as evolutionary dead-ends with little biological relevance. Here we review a range of recent studies that have unveiled a significant number of functional retroposed gene copies in both mammalian and some non-mammalian genomes. These studies have not only revealed previously unknown mechanisms for the emergence of new genes and their functions but have also provided fascinating general insights into molecular and evolutionary processes that have shaped genomes. For example, analyses of chromosomal gene movement patterns via RNA-based gene duplication have shed fresh light on the evolutionary origin and biology of our sex chromosomes.

Circular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise, overall contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires from five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous exonic loci across all species. Even the circRNAs from orthologous loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution – as a byproduct of splicing in orthologs prone to transposon insertion. Overall, our findings argue against widespread functional circRNA conservation.

Only a very small fraction of long noncoding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but the absence of lncRNA annotations in non-model organisms has precluded comparative analyses. Here we present a large-scale evolutionary study of lncRNA repertoires and expression patterns, in 11 tetrapod species. We identify approximately 11,000 primate-specific lncRNAs and 2,500 highly conserved lncRNAs, including approximately 400 genes that are likely to have originated more than 300 million years ago. We find that lncRNAs, in particular ancient ones, are in general actively regulated and may function predominantly in embryonic development. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network suggests potential functions for lncRNAs in fundamental processes such as spermatogenesis and synaptic transmission, but also in more specific mechanisms such as placenta development through microRNA production. Evolutionary study of long noncoding RNA (lncRNA) repertoires and expression patterns in 11 tetrapod species identifies approximately 11,000 primate-specific lncRNAs and 2,500 highly conserved lncRNAs, including approximately 400 genes that are likely to have ancient origins; many lncRNAs, particularly ancient ones, are actively regulated and may function mainly in embryonic development. Regulatory lncRNAs go back a long way Little is known about the evolutionary history of long noncoding RNAs (lncRNAs), but such insight could shed light on their functionality. To this end Henrik Kaessmann and colleagues present the first large-scale evolutionary study of lncRNA repertoires and expression patterns, in eleven tetrapod species. They identify more than 10,000 primate-specific lncRNAs and about 2,500 highly conserved lncRNAs, about 400 of which probably originated at least 300 million years ago, very early in tetrapod history. Many lncRNAs, especially the more ancient ones, are still in active use and may function largely in the regulation of embryonic development as well as other functions from spermatogenesis to synaptic transmission.

Y chromosomes underlie sex determination in mammals, but their repeat-rich nature has hampered sequencing and associated evolutionary studies. Here we trace Y evolution across 15 representative mammals on the basis of high-throughput genome and transcriptome sequencing. We uncover three independent sex chromosome originations in mammals and birds (the outgroup). The original placental and marsupial (therian) Y, containing the sex-determining gene SRY , emerged in the therian ancestor approximately 180 million years ago, in parallel with the first of five monotreme Y chromosomes, carrying the probable sex-determining gene AMH . The avian W chromosome arose approximately 140 million years ago in the bird ancestor. The small Y/W gene repertoires, enriched in regulatory functions, were rapidly defined following stratification (recombination arrest) and erosion events and have remained considerably stable. Despite expression decreases in therians, Y/W genes show notable conservation of proto-sex chromosome expression patterns, although various Y genes evolved testis-specificities through differential regulatory decay. Thus, although some genes evolved novel functions through spatial/temporal expression shifts, most Y genes probably endured, at least initially, because of dosage constraints. Using high-throughput genome and transcriptome sequencing, Y chromosome evolution across 15 representative mammals is explored, with results providing evidence for three independent sex chromosome originations in mammals and birds. Evolution and function of the Y chromosome Mammalian Y chromosomes, known for their roles in sex determination and male fertility, often contain repetitive sequences that make them harder to assemble than the rest of the genome. To counter this problem Henrik Kaessmann and colleagues have developed a new transcript assembly approach based on male-specific RNA/genomic sequencing data to explore Y evolution across 15 species representing all major mammalian lineages. They find evidence for two independent sex chromosome originations in mammals and one in birds. Their analysis of the Y/W gene repertoires suggests that although some genes evolved novel functions in sex determination/spermatogenesis as a result of temporal/spatial expression changes, most Y genes probably persisted, at least initially, as a result of dosage constraints. In a parallel study, Daniel Bellott and colleagues reconstructed the evolution of the Y chromosome, using a comprehensive comparative analysis of the genomic sequence of X–Y gene pairs from seven placental mammals and one marsupial. They conclude that evolution streamlined the gene content of the human Y chromosome through selection to maintain the ancestral dosage of homologous X–Y gene pairs that regulate gene expression throughout the body. They propose that these genes make the Y chromosome essential for male viability and contribute to differences between the sexes in health and disease.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer 1 . Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer—potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs. An analysis using ribosome-profiling and matched RNA-sequencing data for three organs across five mammalian species and a bird enables the comparison of translatomes and transcriptomes, revealing patterns of co-evolution of these two expression layers.

Promoters and enhancers—key controllers of gene expression—have long been distinguished from each other based on their function. However, recent work suggested that common architectural and functional features might have facilitated the conversion of one type of element into the other during evolution. Here, based on cross-mammalian analyses of epigenome and transcriptome data, we provide support for this hypothesis by detecting 445 regulatory elements with signatures of activity turnover (termed P/E elements). Most events represent transformations of putative ancestral enhancers into promoters, leading to the emergence of species-specific transcribed loci or 5′ exons. Distinct GC sequence compositions and stabilizing 5′ splicing (U1) regulatory motif patterns may have predisposed P/E elements to regulatory repurposing, and changes in the U1 and polyadenylation signal densities and distributions likely drove the evolutionary activity switches. Our work suggests that regulatory repurposing facilitated regulatory innovation and the origination of new genes and exons during evolution. Enhancers and promoters are different types of regulatory elements with shared architectural and functional features. Here the authors perform integrated cross-mammalian analyses of DNase hypersensitivity, chromatin modification and transcriptional data, to provide evidence of regulatory repurposing during evolution.

Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals. Gene expression and species difference Genome analyses can uncover protein-coding changes that potentially underlie the differences between species, but many of the phenotypic differences between species are the result of regulatory mutations affecting gene expression. Brawand et al . use high-throughput RNA sequencing to study the evolutionary dynamics of mammalian transcriptomes in six major tissues (cortex, cerebellum, heart, kidney, liver and testis) from ten species from all major mammalian lineages. Among the findings is the extent of transcriptome variation between organs and species, as well as the identification of potentially selectively driven expression switches that may have shaped specific organ biology.

Ionotropic glutamate receptors (iGluRs) are a highly conserved family of ligand-gated ion channels present in animals, plants, and bacteria, which are best characterized for their roles in synaptic communication in vertebrate nervous systems. A variant subfamily of iGluRs, the Ionotropic Receptors (IRs), was recently identified as a new class of olfactory receptors in the fruit fly, Drosophila melanogaster, hinting at a broader function of this ion channel family in detection of environmental, as well as intercellular, chemical signals. Here, we investigate the origin and evolution of IRs by comprehensive evolutionary genomics and in situ expression analysis. In marked contrast to the insect-specific Odorant Receptor family, we show that IRs are expressed in olfactory organs across Protostomia--a major branch of the animal kingdom that encompasses arthropods, nematodes, and molluscs--indicating that they represent an ancestral protostome chemosensory receptor family. Two subfamilies of IRs are distinguished: conserved \"antennal IRs,\" which likely define the first olfactory receptor family of insects, and species-specific \"divergent IRs,\" which are expressed in peripheral and internal gustatory neurons, implicating this family in taste and food assessment. Comparative analysis of drosophilid IRs reveals the selective forces that have shaped the repertoires in flies with distinct chemosensory preferences. Examination of IR gene structure and genomic distribution suggests both non-allelic homologous recombination and retroposition contributed to the expansion of this multigene family. Together, these findings lay a foundation for functional analysis of these receptors in both neurobiological and evolutionary studies. Furthermore, this work identifies novel targets for manipulating chemosensory-driven behaviours of agricultural pests and disease vectors.