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Here we describe a new integrative approach for accurate annotation and quantification of circRNAs named Short Read circRNA Pipeline (SRCP). Our strategy involves two steps: annotation of validated circRNAs followed by a quantification step. We show that SRCP is more sensitive than other individual pipelines and allows for more comprehensive quantification of a larger number of differentially expressed circRNAs. To facilitate the use of SRCP, we generate a comprehensive collection of validated circRNAs in five different organisms, including humans. We then utilize our approach and identify a subset of circRNAs bound to the miRNA-effector protein AGO2 in human brain samples. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-021-02497-7.

Precision medicine and sequence-based clinical diagnostics seek to predict disease risk or to identify causative variants from sequencing data. The Critical Assessment of Genome Interpretation (CAGI) is a community experiment consisting of genotype-phenotype prediction challenges; participants build models, undergo assessment, and share key findings. In the past, few CAGI challenges have addressed the impact of sequence variants on splicing. In CAGI5, two challenges (Vex-seq and MaPSY) involved prediction of the effect of variants, primarily single-nucleotide changes, on splicing. Although there are significant differences between these two challenges, both involved prediction of results from high-throughput exon inclusion assays. Here, we discuss the methods used to predict the impact of these variants on splicing, their performance, strengths, and weaknesses, and prospects for predicting the impact of sequence variation on splicing and disease phenotypes.

Changes in potential regulatory elements are thought to be key drivers of phenotypic divergence. However, identifying changes to regulatory elements that underlie human-specific traits has proven very challenging. Here, we use 63 reconstructed and experimentally measured DNA methylation maps of ancient and present-day humans, as well as of six chimpanzees, to detect differentially methylated regions that likely emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes associated with face and vocal tract anatomy went through particularly extensive methylation changes. Specifically, we identify widespread hypermethylation in a network of face- and voice-associated genes (SOX9, ACAN, COL2A1, NFIX and XYLT1). We propose that these repression patterns appeared after the split from Neanderthals and Denisovans, and that they might have played a key role in shaping the modern human face and vocal tract., D.G. is supported by the Clore Israel Foundation. TMB is supported by BFU2017-86471-P (MINECO/FEDER, UE), U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca and CERCA Program del Departament d’Economia i Coneixement de la Generalitat de Catalunya. D.R. is an Investigator of the Howard Hughes Medical Institute and is also supported by an Allen Discovery Center for the Study of Human Brain Evolution funded the Paul G. Allen Family Foundation. C.L.-F. is supported by FEDER and BFU2015-64699-P grant from the Spanish government. R.P. was supported by ERC starting grant ADNABIOARC (263441). R.M.G. and J.M.O. are supported by NYSTEM contract C030133. Funding for the collection and processing of the 850K chimpanzee data was provided by the Leakey Foundation Research Grant for Doctoral Students, Wenner-Gren Foundation Dissertation Fieldwork Grant (Gr. 9310), James F. Nacey Fellowship from the Nacey Maggioncalda Foundation, International Primatological Society Research Grant, Sigma Xi Grant-in-Aid of Research, Center for Evolution and Medicine Venture Fund (ASU), Graduate Research and Support Program Grant (GPSA, ASU), and Graduate Student Research Grant (SHESC, ASU) to G.H. Collection of the chimpanzee bone from Tanzania was funded by the Jane Goodall Institute, and grants from the US National Institutes of Health (AI 058715) and National Science Foundation (IOS-1052693), and facilitated by Elizabeth Lonsdorf and Beatrice Hahn.

Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3′-untranslated regions (3′-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3′-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3′-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20–25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

Analyzing the conditions in which past individuals lived is key to understanding the environments and cultural transitions to which humans had to adapt. Here, we suggest a methodology to probe into past environments, using reconstructed premortem DNA methylation maps of ancient individuals. We review a large body of research showing that differential DNA methylation is associated with changes in various external and internal factors, and propose that loci whose DNA methylation level is environmentally responsive could serve as markers to infer about ancient daily life, diseases, nutrition, exposure to toxins, and more. We demonstrate this approach by showing that hunger-related DNA methylation changes are found in ancient hunter-gatherers. The strategy we present here opens a window to reconstruct previously inaccessible aspects of the lives of past individuals.

Many human introns carry out a function, in the sense that they are critical to maintain normal cellular activity. Their identification is fundamental to understanding cellular processes and disease. However, being noncoding elements, such functional introns are poorly predicted based on traditional approaches of sequence and structure conservation. Here, we generated a dataset of human functional introns that carry out different types of functions. We showed that functional introns share common characteristics, such as higher positional conservation along the coding sequence and reduced loss rates, regardless of their specific function. A unique property of the data is that if an intron is unknown to be functional, it still does not mean that it is indeed non-functional. We developed a probabilistic framework that explicitly accounts for this unique property, and predicts which specific human introns are functional. We show that we successfully predict function even when the algorithm is trained on introns with a different type of function. This ability has many implications in studying regulatory networks, gene regulation, the effect of mutations outside exons on human disease, and on our general understanding of intron evolution and their functional exaptation in mammals.

DNA methylation maps can be used to predict anatomical features in hominins and chimpanzees, allowing for reconstruction of a putative anatomical profile of the Denisovan, currently absent from the fossil record.Denisovans are an extinct group of humans whose morphology remains unknown. Here, we present a method for reconstructing skeletal morphology using DNA methylation patterns. Our method is based on linking unidirectional methylation changes to loss-of-function phenotypes. We tested performance by reconstructing Neanderthal and chimpanzee skeletal morphologies and obtained >85% precision in identifying divergent traits. We then applied this method to the Denisovan and offer a putative morphological profile. We suggest that Denisovans likely shared with Neanderthals traits such as an elongated face and a wide pelvis. We also identify Denisovan-derived changes, such as an increased dental arch and lateral cranial expansion. Our predictions match the only morphologically informative Denisovan bone to date, as well as the Xuchang skull, which was suggested by some to be a Denisovan. We conclude that DNA methylation can be used to reconstruct anatomical features, including some that do not survive in the fossil record., This work has been supported by the National Geographic Society (grant HJ-111R-17 to L.C.). D.G. was supported by the Clore Israel Foundation. T.M.-B. is supported by BFU2017-86471-P (MINECO/FEDER, UE), U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa,” and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880).

An appreciable fraction of introns is thought to have some function, but there is no obvious way to predict which specific intron is likely to be functional. We hypothesize that functional introns experience a different selection regime than non-functional ones and will therefore show distinct evolutionary histories. In particular, we expect functional introns to be more resistant to loss, and that this would be reflected in high conservation of their position with respect to the coding sequence. To test this hypothesis, we focused on introns whose function comes about from microRNAs and snoRNAs that are embedded within their sequence. We built a data set of orthologous genes across 28 eukaryotic species, reconstructed the evolutionary histories of their introns and compared functional introns with the rest of the introns. We found that, indeed, the position of microRNA- and snoRNA-bearing introns is significantly more conserved. In addition, we found that both families of RNA genes settled within introns early during metazoan evolution. We identified several easily computable intronic properties that can be used to detect functional introns in general, thereby suggesting a new strategy to pinpoint non-coding cellular functions.

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.

SummaryTo realize the promise of stem cell biology, it is important to identify the precise time in the history of the cell when developmental potential is restricted. To achieve this goal, we developed a real-time imaging system that captures the transitions in fate, generating neurons, astrocytes, and oligodendrocytes from single CNS stem cells in vitro. In the presence of bFGF, tripotent cells normally produce specified progenitors through a bipotent intermediate cell type. Surprisingly, the tripotent state is reset at each passage. The cytokine CNTF is thought to instruct multipotent cells to an astrocytic fate. We demonstrate that CNTF both directs astrogliogenesis from tripotent cells, bypassing two of the three normal bipotent intermediates, and later promotes the expansion of specified astrocytic progenitors. These results show how discrete cell types emerge from a multipotent cell and provide a strong basis for future studies to determine the molecular basis of fate specification.

Numerous eukaryotic proteins contain multiple domains. Certain domains show a tendency to occur in diverse domain architectures and can be considered “promiscuous.” These promiscuous domains are, typically, involved in protein–protein interactions and play crucial roles in interaction networks, particularly those that contribute to signal transduction. A systematic comparative-genomic analysis of promiscuous domains in eukaryotes is described. Two quantitative measures of domain promiscuity are introduced and applied to the analysis of 28 genomes of diverse eukaryotes. Altogether, 215 domains are identified as strongly promiscuous. The fraction of promiscuous domains in animals is shown to be significantly greater than that in fungi or plants. Evolutionary reconstructions indicate that domain promiscuity is a volatile, relatively fast-changing feature of eukaryotic proteins, with few domains remaining promiscuous throughout the evolution of eukaryotes. Some domains appear to have attained promiscuity independently in different lineages, for example, animals and plants. It is proposed that promiscuous domains persist within a relatively small pool of evolutionarily stable domain combinations from which numerous rare architectures emerge during evolution. Domain promiscuity positively correlates with the number of experimentally detected domain interactions and with the strength of purifying selection affecting a domain. Thus, evolution of promiscuous domains seems to be constrained by the diversity of their interaction partners. The set of promiscuous domains is enriched for domains mediating protein–protein interactions that are involved in various forms of signal transduction, especially in the ubiquitin system and in chromatin. Thus, a limited repertoire of promiscuous domains makes a major contribution to the diversity and evolvability of eukaryotic proteomes and signaling networks.

Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.

Introns that interrupt eukaryotic protein-coding sequences are generally thought to be nonfunctional. However, for reasons still poorly understood, positions of many introns are highly conserved in evolution. Previous reconstructions of intron gain and loss events during eukaryotic evolution used a variety of simplified evolutionary models that yielded contradicting conclusions and are not suited to reveal some of the key underlying processes. We combine a comprehensive probabilistic model and an extended data set, including 391 conserved genes from 19 eukaryotes, to uncover previously unnoticed aspects of intron evolution—in particular, to assign intron gain and loss rates to individual genes. The rates of intron gain and loss in a gene show moderate positive correlation. A gene’s intron gain rate shows a highly significant negative correlation with the coding-sequence evolution rate; intron loss rate also significantly, but positively, correlates with the sequence evolution rate. Correlations of the opposite signs, albeit less significant ones, are observed between intron gain and loss rates and gene expression level. It is proposed that intron evolution includes a neutral component, which is manifest in the positive correlation between the gain and loss rates and a selection-driven component as reflected in the links between intron gain and loss and sequence evolution. The increased intron gain and decreased intron loss in evolutionarily conserved genes indicate that intron insertion often might be adaptive, whereas some of the intron losses might be deleterious. This apparent functional importance of introns is likely to be due, at least in part, to their multiple effects on gene expression.

Recent years have witnessed the rise of ancient DNA (aDNA) technology, allowing comparative genomics to be carried out at unprecedented time resolution. While it is relatively straightforward to use aDNA to identify recent genomic changes, it is much less clear how to utilize it to study changes in epigenetic regulation. Here we review recent works demonstrating that highly degraded aDNA still contains sufficient information to allow reconstruction of epigenetic signals, including DNA methylation and nucleosome positioning maps. We discuss challenges arising from the tissue specificity of epigenetics, and show how some of them might in fact turn into advantages. Finally, we introduce a method to infer methylation states in tissues that do not tend to be preserved over time.

SummaryThe T cell receptor (TCR) controls the cellular adaptive immune response to antigens, but our understanding of TCR repertoire diversity and response to challenge is still incomplete. For example, TCR clones shared by different individuals with minimal alteration to germline gene sequences (public clones) are detectable in all vertebrates, but their significance is unknown. Although small in size, the zebrafish TCR repertoire is controlled by processes similar to those operating in mammals. Thus, we studied the zebrafish TCR repertoire and its response to stimulation with self and foreign antigens. We found that cross-reactive public TCRs dominate the T cell response, endowing a limited TCR repertoire with the ability to cope with diverse antigenic challenges. These features of vertebrate public TCRs might provide a mechanism for the rapid generation of protective T cell immunity, allowing a short temporal window for the development of more specific private T cell responses.

Methylating the Family Tree DNA sequences show a high level of similarities between humans and ancient hominids but the degree to which there are differences between methylated regions in their genomes that may explain phenotypic differences is unclear. Gokhman et al. (p. 523 , published online 17 April) demonstrate that naturally degraded methylated cytosines in ancient DNA are converted to thymines and can be used to reconstruct ancient methylomes. The results suggest differences in methylation in bone tissues between modern humans and ancient hominids in a set of genes important for limb development.

A recent study conducted the first genome-wide scan for selection in Inuit from Greenland using SNP chip data. Here, we report that selection in the region with the second most extreme signal of positive selection in Greenlandic Inuit favored a deeply divergent haplotype that is closely related to the sequence in the Denisovan genome, and was likely introgressed from an archaic population. The region contains two genes,WARS2andTBX15, and has previously been associated with adipose tissue differentiation and body-fat distribution in humans. We show that the adaptively introgressed allele has been under selection in a much larger geographic region than just Greenland. Furthermore, it is associated with changes in expression ofWARS2andTBX15in multiple tissues including the adrenal gland and subcutaneous adipose tissue, and with regional DNA methylation changes inTBX15.

Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers’ Reports section.

Intron density is highly variable across eukaryotic species. It seems that different lineages have experienced considerably different levels of intron gain and loss events, but the reasons for this are not well known. A large number of mechanisms for intron loss and gain have been suggested, and most of them have at least some level of indirect support. We therefore figured out that the variability in intron density can be a reflection of the fact that different mechanisms are active in different lineages. Quite a number of these putative mechanisms, both for intron loss and for intron gain, postulate that the enzyme reverse transcriptase (RT) has a key role in the process. In this paper, we lay out three predictions whose approval or falsification gives indication for the involvement of RT in intron gain and loss processes. Testing these predictions requires data on the intron gain and loss rates of individual genes along different branches of the eukaryotic phylogenetic tree. So far, such rates could not be computed, and hence, these predictions could not be rigorously evaluated. Here, we use a maximum likelihood algorithm that we have devised in the past, Evolutionary Reconstruction by Expectation Maximization, which allows the estimation of such rates. Using this algorithm, we computed the intron loss and gain rates of more than 300 genes in each branch of the phylogenetic tree of 19 eukaryotic species. Based on that we found only little support for RT activity in intron gain. In contrast, we suggest that RT-mediated intron loss is a mechanism that is very efficient in removing introns, and thus, its levels of activity may be a major determinant of intron number. Moreover, we found that intron gain and loss rates are negatively correlated in intron-poor species but are positively correlated for intron-rich species. One explanation to this is that intron gain and loss mechanisms in intron-rich species (like metazoans) share a common mechanistic component, albeit not a RT.

Background Rare genomic changes (RGCs) that are thought to comprise derived shared characters of individual clades are becoming an increasingly important class of markers in genome-wide phylogenetic studies. Recently, we proposed a new type of RGCs designated RGC_CAMs (after Conserved Amino acids-Multiple substitutions) that were inferred using genome-wide identification of amino acid replacements that were: i) located in unambiguously aligned regions of orthologous genes, ii) shared by two or more taxa in positions that contain a different, conserved amino acid in a much broader range of taxa, and iii) require two or three nucleotide substitutions. When applied to animal phylogeny, the RGC_CAM approach supported the coelomate clade that unites deuterostomes with arthropods as opposed to the ecdysozoan (molting animals) clade. However, a non-negligible level of homoplasy was detected. Results We provide a direct estimate of the level of homoplasy caused by parallel changes and reversals among the RGC_CAMs using 462 alignments of orthologous genes from 19 eukaryotic species. It is shown that the impact of parallel changes and reversals on the results of phylogenetic inference using RGC_CAMs cannot explain the observed support for the Coelomata clade. In contrast, the evidence in support of the Ecdysozoa clade, in large part, can be attributed to parallel changes. It is demonstrated that parallel changes are significantly more common in internal branches of different subtrees that are separated from the respective common ancestor by relatively short times than in terminal branches separated by longer time intervals. A similar but much weaker trend was detected for reversals. The observed evolutionary trend of parallel changes is explained in terms of the covarion model of molecular evolution. As the overlap between the covarion sets in orthologous genes from different lineages decreases with time after divergence, the likelihood of parallel changes decreases as well. Conclusion The level of homoplasy observed here appears to be low enough to justify the utility of RGC_CAMs and other types of RGCs for resolution of hard problems in phylogeny. Parallel changes, one of the major classes of events leading to homoplasy, occur much more often in relatively recently diverged lineages than in those separated from their last common ancestor by longer time intervals of time. This pattern seems to provide the molecular-evolutionary underpinning of Vavilov's law of homologous series and is readily interpreted within the framework of the covarion model of molecular evolution. Reviewers This article was reviewed by Alex Kondrashov, Nicolas Galtier, and Maximilian Telford and Robert Lanfear (nominated by Laurence Hurst).

The recent analysis of a novel class of rare genomic changes, RGC_CAMs (after conserved amino acids-multiple substitutions), supported the Coelomata clade of animals as opposed to the Ecdysozoa clade (Rogozin et al. 2007). A subsequent reanalysis, with the sequences from the sea anemone Nematostella vectensis included in the set of outgroup species, suggested that this result was an artifact caused by reverse amino replacements and claimed support for Ecdysozoa (Irimia et al. 2007). We show that the internal branch connecting the sea anemone to the bilaterian animals is extremely short, resulting in a weak statistical support for the Coelomata clade. Direct estimation of the level of homoplasy, combined with taxon sampling with different sets of outgroup species, reinforces the support for Coelomata, whereas the effect of reversals is shown to be relatively minor.

Several contrasting scenarios have been proposed for the origin and evolution of spliceosomal introns, a hallmark of eukaryotic genes. A comprehensive probabilistic model to obtain a definitive reconstruction of intron evolution was developed and applied to 391 sets of conserved genes from 19 eukaryotic species. It is inferred that a relatively high intron density was reached early, i.e., the last common ancestor of eukaryotes contained >2.15 introns/kilobase, and the last common ancestor of multicellular life forms harbored approximately 3.4 introns/kilobase, a greater intron density than in most of the extant fungi and in some animals. The rates of intron gain and intron loss appear to have been dropping during the last approximately 1.3 billion years, with the decline in the gain rate being much steeper. Eukaryotic lineages exhibit three distinct modes of evolution of the intron-exon structure. The primary, balanced mode, apparently, operates in all lineages. In this mode, intron gain and loss are strongly and positively correlated, in contrast to previous reports on inverse correlation between these processes. The second mode involves an elevated rate of intron loss and is prevalent in several lineages, such as fungi and insects. The third mode, characterized by elevated rate of intron gain, is seen only in deep branches of the tree, indicating that bursts of intron invasion occurred at key points in eukaryotic evolution, such as the origin of animals. Intron dynamics could depend on multiple mechanisms, and in the balanced mode, gain and loss of introns might share common mechanistic features.

As the number of sequenced genomes from diverse walks of life rapidly increases, phylogenetic analysis is entering a new era: reconstruction of the evolutionary history of organisms on the basis of full-scale comparison of their genomes. In addition to brute force, genome-wide analysis of alignments, rare genomic changes (RGCs) that are thought to comprise derived shared characters of individual clades are increasingly used in genome-wide phylogenetic studies. We propose a new type of RGCs designated RGC_CAMs (after Conserved Amino acids-Multiple substitutions), which are inferred using a genome-scale analysis of protein and underlying nucleotide sequence alignments. The RGC_CAM approach utilizes amino acid residues conserved in major eukaryotic lineages, with the exception of a few species comprising a putative clade, and selects for phylogenetic inference only those amino acid replacements that require 2 or 3 nucleotide substitutions, in order to reduce homoplasy. The RGC_CAM analysis was combined with a procedure for rigorous statistical testing of competing phylogenetic hypotheses. The RGC_CAM method is shown to be robust to branch length differences and taxon sampling. When applied to animal phylogeny, the RGC_CAM approach strongly supports the coelomate clade that unites chordates with arthropods as opposed to the ecdysozoan (molting animals) clade. This conclusion runs against the view of animal evolution that is currently prevailing in the evo-devo community. The final solution to the coelomate-ecdysozoa controversy will require a much larger set of complete genome sequences representing diverse animal taxa. It is expected that RGC_CAM and other RGC-based methods will be crucial for these future, definitive phylogenetic studies.

Recent genome analyses revealed intriguing correlations between variables characterizing the functioning of a gene, such as expression level (EL), connectivity of genetic and protein–protein interaction networks, and knockout effect, and variables describing gene evolution, such as sequence evolution rate (ER) and propensity for gene loss. Typically, variables within each of these classes are positively correlated, e.g. products of highly expressed genes also have a propensity to be involved in many protein–protein interactions, whereas variables between classes are negatively correlated, e.g. highly expressed genes, on average, evolve slower than weakly expressed genes. Here, we describe principal component (PC) analysis of seven genome-related variables and propose biological interpretations for the first three PCs. The first PC reflects a gene's ‘importance’, or the ‘status’ of a gene in the genomic community, with positive contributions from knockout lethality, EL, number of protein–protein interaction partners and the number of paralogues, and negative contributions from sequence ER and gene loss propensity. The next two PCs define a plane that seems to reflect the functional and evolutionary plasticity of a gene. Specifically, PC2 can be interpreted as a gene's ‘adaptability’ whereby genes with high adaptability readily duplicate, have many genetic interaction partners and tend to be non-essential. PC3 also might reflect the role of a gene in organismal adaptation albeit with a negative rather than a positive contribution of genetic interactions; we provisionally designate this PC ‘reactivity’. The interpretation of PC2 and PC3 as measures of a gene's plasticity is compatible with the observation that genes with high values of these PCs tend to be expressed in a condition- or tissue-specific manner. Functional classes of genes substantially vary in status, adaptability and reactivity, with the highest status characteristic of the translation system and cytoskeletal proteins, highest adaptability seen in cellular processes and signalling genes, and top reactivity characteristic of metabolic enzymes.

Although the family of genes encoding for olfactory receptors was identified more than 15 years ago, the difficulty of functionally expressing these receptors in an heterologous system has, with only some exceptions, rendered the receptive range of given olfactory receptors largely unknown. Furthermore, even when successfully expressed, the task of probing such a receptor with thousands of odors/ligands remains daunting. Here we provide proof of concept for a solution to this problem. Using computational methods, we tune an electronic nose to the receptive range of an olfactory receptor. We then use this electronic nose to predict the receptors' response to other odorants. Our method can be used to identify the receptive range of olfactory receptors, and can also be applied to other questions involving receptor–ligand interactions in non-olfactory settings., Author Summary A key goal in biology is to identify specific ligands for specific receptors. One example is where the ligand is a drug. In turn, in the olfactory system the ligand is the odorant that binds to olfactory receptors. There are many olfactory receptor types, and which odorants will activate which receptors remains largely unknown. One way to answer this is to systematically vary the molecular features of ligands and to measure the olfactory receptor response. However, the vast number of molecular features and their combinations renders such an effort potentially unsolvable. Here, rather than looking at the trees (each molecular feature), we looked at the forest (the smell they generate). We used a device called an electronic nose that generates a patterned response to odorants. We then obtained the response to a set of odorants that are known to activate a particular olfactory receptor, and we used this pattern to predict the response of that receptor to other odorants. We found that, on average in three out of four we could predict the response of olfactory receptors. This result provides a new method for probing the olfactory system, and also suggests a novel method for identifying potential drugs.