Evolution of tissue-specific expression of ancestral genes across vertebrates and insects.

Regulation of gene expression is arguably the main mechanism underlying the phenotypic diversity of tissues within and between species. Here we assembled an extensive transcriptomic dataset covering 8 tissues across 20 bilaterian species and performed analyses using a symmetric phylogeny that allowed the combined and parallel investigation of gene expression evolution between vertebrates and insects. We specifically focused on widely conserved ancestral genes, identifying strong cores of pan-bilaterian tissue-specific genes and even larger groups that diverged to define vertebrate and insect tissues. Systematic inferences of tissue-specificity gains and losses show that nearly half of all ancestral genes have been recruited into tissue-specific transcriptomes. This occurred during both ancient and, especially, recent bilaterian evolution, with several gains being associated with the emergence of unique phenotypes (for example, novel cell types). Such pervasive evolution of tissue specificity was linked to gene duplication coupled with expression specialization of one of the copies, revealing an unappreciated prolonged effect of whole-genome duplications on recent vertebrate evolution.

Genes enriched in A/T-ending codons are co-regulated and conserved across mammals.

Codon usage influences gene expression distinctly depending on the cell context. Yet, the importance of codon bias in the simultaneous turnover of specific groups of protein-coding genes remains to be investigated. Here, we find that genes enriched in A/T-ending codons are expressed more coordinately in general and across tissues and development than those enriched in G/C-ending codons. tRNA abundance measurements indicate that this coordination is linked to the expression changes of tRNA isoacceptors reading A/T-ending codons. Genes with similar codon composition are more likely to be part of the same protein complex, especially for genes with A/T-ending codons. The codon preferences of genes with A/T-ending codons are conserved among mammals and other vertebrates. We suggest that this orchestration contributes to tissue-specific and ontogenetic-specific expression, which can facilitate, for instance, timely protein complex formation.

Parallel evolution of amphioxus and vertebrate small-scale gene duplications.

BACKGROUND: Amphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates. RESULTS: We present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements. CONCLUSIONS: In contrast to their slower molecular and morphological evolution, amphioxus' small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms.

The 3D-Evo Space: Evolution of Gene Expression and Alternative Splicing Regulation.

Animal species present relatively high levels of gene conservation, and yet they display a great variety of cell type and tissue phenotypes. These diverse phenotypes are mainly specified through differential gene usage, which relies on several mechanisms. Two of the most relevant mechanisms are regulated gene transcription, usually referred to as gene expression (rGE), and regulated alternative splicing (rAS). Several works have addressed how either rGE or rAS contributes to phenotypic diversity throughout evolution, but a back-to-back comparison between the two molecular mechanisms, specifically highlighting both their common regulatory principles and unique properties, is still missing. In this review, we propose an innovative framework for the unified comparison between rGE and rAS from different perspectives: the three-dimensional (3D)-evo space. We use the 3D-evo space to comprehensively (a) review the molecular basis of rGE and rAS (i.e., the molecular axis), (b) depict the tissue-specific phenotypes they contribute to (i.e., the tissue axis), and (c) describe the determinants that drive the evolution of rGE and rAS programs (i.e., the evolution axis). Finally, we unify the perspectives emerging from the three axes by discussing general trends and specific examples of rGE and rAS tissue program evolution.

Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

Genome-wide transcriptomic changes reveal the genetic pathways involved in insect migration.

Insects are capable of extraordinary feats of long-distance movement that have profound impacts on the function of terrestrial ecosystems. The ability to undertake these movements arose multiple times through the evolution of a suite of traits that make up the migratory syndrome, however the underlying genetic pathways involved remain poorly understood. Migratory hoverflies (Diptera: Syrphidae) are an emerging model group for studies of migration. They undertake seasonal movements in huge numbers across large parts of the globe and are important pollinators, biological control agents and decomposers. Here, we assembled a high-quality draft genome of the marmalade hoverfly (Episyrphus balteatus). We leveraged this genomic resource to undertake a genome-wide transcriptomic comparison of actively migrating Episyrphus, captured from a high mountain pass as they flew south to overwinter, with the transcriptomes of summer forms which were non-migratory. We identified 1543 genes with very strong evidence for differential expression. Interrogation of this gene set reveals a remarkable range of roles in metabolism, muscle structure and function, hormonal regulation, immunity, stress resistance, flight and feeding behaviour, longevity, reproductive diapause and sensory perception. These features of the migrant phenotype have arisen by the integration and modification of pathways such as insulin signalling for diapause and longevity, JAK/SAT for immunity, and those leading to octopamine production and fuelling to boost flight capabilities. Our results provide a powerful genomic resource for future research, and paint a comprehensive picture of global expression changes in an actively migrating insect, identifying key genomic components involved in this important life-history strategy.

Computational Analysis of Alternative Splicing Using VAST-TOOLS and the VastDB Framework.

Alternative splicing (AS) can vastly expand animal transcriptomes and proteomes. Two main open questions in the field are how AS is regulated across cell/tissue types and disease, and what roles different AS events play. To facilitate AS research, we have created the computational VastDB framework, which comprises a series of complementary software and resources that we describe in this chapter. The VastDB framework is especially designed to aid biomedical researchers without a strong computational background. It offers tools and resources to: (a) quantify AS and identify differentially spliced AS events using RNA-seq data (vast-tools), (b) perform multiple genomic and sequence analyses for investigating AS events (Matt), (c) identify AS events with genomic and regulatory conservation among species (ExOrthist), and (d) help with the biological interpretation of the results, and, ultimately, with the identification of interesting AS events to design wet-lab experiments (VastDB and PastDB).

Specialization of the photoreceptor transcriptome by Srrm3-dependent microexons is required for outer segment maintenance and vision.

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.

ExOrthist: a tool to infer exon orthologies at any evolutionary distance.

Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes. However, no corresponding tool allows the detection of exon homology relationships. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling inference of exon homologs and orthogroups, visualization of evolution of exon-intron structures, and assessment of conservation of alternative splicing patterns. ExOrthist evaluates exon sequence conservation and considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in different evolutionary scenarios: whole genome duplication in frogs and convergence of Nova-regulated splicing networks ( https://github.com/biocorecrg/ExOrthist ).

Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals.

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.

Evolutionary Rewiring of Human Regulatory Networks by Waves of Genome Expansion.

Genome expansion is believed to be an important driver of the evolution of gene regulation. To investigate the role of a newly arising sequence in rewiring regulatory networks, we estimated the age of each region of the human genome by applying maximum parsimony to genome-wide alignments with 100 vertebrates. We then studied the age distribution of several types of functional regions, with a focus on regulatory elements. The age distribution of regulatory elements reveals the extensive use of newly formed genomic sequence in the evolution of regulatory interactions. Many transcription factors have expanded their repertoire of targets through waves of genomic expansions that can be traced to specific evolutionary times. Repeated elements contributed a major part of such expansion: many classes of such elements are enriched in binding sites of one or a few specific transcription factors, whose binding sites are localized in specific portions of the element and characterized by distinctive motif words. These features suggest that the binding sites were available as soon as the new sequence entered the genome, rather than being created later by accumulation of point mutations. By comparing the age of regulatory regions to the evolutionary shift in expression of nearby genes, we show that rewiring through genome expansion played an important role in shaping human regulatory networks.