Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Resurrection of endogenous retroviruses during aging reinforces senescence.

Whether and how certain transposable elements with viral origins, such as endogenous retroviruses (ERVs) dormant in our genomes, can become awakened and contribute to the aging process is largely unknown. In human senescent cells, we found that HERVK (HML-2), the most recently integrated human ERVs, are unlocked to transcribe viral genes and produce retrovirus-like particles (RVLPs). These HERVK RVLPs constitute a transmissible message to elicit senescence phenotypes in young cells, which can be blocked by neutralizing antibodies. The activation of ERVs was also observed in organs of aged primates and mice as well as in human tissues and serum from the elderly. Their repression alleviates cellular senescence and tissue degeneration and, to some extent, organismal aging. These findings indicate that the resurrection of ERVs is a hallmark and driving force of cellular senescence and tissue aging.

Adaptive evolution of the enigmatic Takakia now facing climate change in Tibet.

The most extreme environments are the most vulnerable to transformation under a rapidly changing climate. These ecosystems harbor some of the most specialized species, which will likely suffer the highest extinction rates. We document the steepest temperature increase (2010-2021) on record at altitudes of above 4,000 m, triggering a decline of the relictual and highly adapted moss Takakia lepidozioides. Its de-novo-sequenced genome with 27,467 protein-coding genes includes distinct adaptations to abiotic stresses and comprises the largest number of fast-evolving genes under positive selection. The uplift of the study site in the last 65 million years has resulted in life-threatening UV-B radiation and drastically reduced temperatures, and we detected several of the molecular adaptations of Takakia to these environmental changes. Surprisingly, specific morphological features likely occurred earlier than 165 mya in much warmer environments. Following nearly 400 million years of evolution and resilience, this species is now facing extinction.

Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

Forces driving transposable element load variation during Arabidopsis range expansion.

Genetic load refers to the accumulated and potentially life-threatening deleterious mutations in populations. Understanding the mechanisms underlying genetic load variation of transposable element (TE) insertion, a major large-effect mutation, during range expansion is an intriguing question in biology. Here, we used 1,115 global natural accessions of Arabidopsis (Arabidopsis thaliana) to study the driving forces of TE load variation during its range expansion. TE load increased with range expansion, especially in the recently established Yangtze River basin population. Effective population size, which explains 62.0% of the variance in TE load, high transposition rate, and selective sweeps contributed to TE accumulation in the expanded populations. We genetically mapped and identified multiple candidate causal genes and TEs, and revealed the genetic architecture of TE load variation. Overall, this study reveals the variation in TE genetic load during Arabidopsis expansion and highlights the causes of TE load variation from the perspectives of both population genetics and quantitative genetics.

Gene expression across mammalian organ development.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

The mutational load in natural populations is significantly affected by high primary rates of retroposition.

Gene retroposition is known to contribute to patterns of gene evolution and adaptations. However, possible negative effects of gene retroposition remain largely unexplored since most previous studies have focused on between-species comparisons where negatively selected copies are mostly not observed, as they are quickly lost from populations. Here, we show for natural house mouse populations that the primary rate of retroposition is orders of magnitude higher than the long-term rate. Comparisons with single-nucleotide polymorphism distribution patterns in the same populations show that most retroposition events are deleterious. Transcriptomic profiling analysis shows that new retroposed copies become easily subject to transcription and have an influence on the expression levels of their parental genes, especially when transcribed in the antisense direction. Our results imply that the impact of retroposition on the mutational load has been highly underestimated in natural populations. This has additional implications for strategies of disease allele detection in humans.

Genomic analyses of new genes and their phenotypic effects reveal rapid evolution of essential functions in Drosophila development.

It is a conventionally held dogma that the genetic basis underlying development is conserved in a long evolutionary time scale. Ample experiments based on mutational, biochemical, functional, and complementary knockdown/knockout approaches have revealed the unexpectedly important role of recently evolved new genes in the development of Drosophila. The recent progress in the genome-wide experimental testing of gene effects and improvements in the computational identification of new genes (< 40 million years ago, Mya) open the door to investigate the evolution of gene essentiality with a phylogenetically high resolution. These advancements also raised interesting issues in techniques and concepts related to phenotypic effect analyses of genes, particularly of those that recently originated. Here we reported our analyses of these issues, including reproducibility and efficiency of knockdown experiment and difference between RNAi libraries in the knockdown efficiency and testing of phenotypic effects. We further analyzed a large data from knockdowns of 11,354 genes (~75% of the Drosophila melanogaster total genes), including 702 new genes (~66% of the species total new genes that aged < 40 Mya), revealing a similarly high proportion (~32.2%) of essential genes that originated in various Sophophora subgenus lineages and distant ancestors beyond the Drosophila genus. The transcriptional compensation effect from CRISPR knockout were detected for highly similar duplicate copies. Knockout of a few young genes detected analogous essentiality in various functions in development. Taken together, our experimental and computational analyses provide valuable data for detection of phenotypic effects of genes in general and further strong evidence for the concept that new genes in Drosophila quickly evolved essential functions in viability during development.

The evolution of ancestral and species-specific adaptations in snowfinches at the Qinghai-Tibet Plateau.

Species in a shared environment tend to evolve similar adaptations under the influence of their phylogenetic context. Using snowfinches, a monophyletic group of passerine birds (Passeridae), we study the relative roles of ancestral and species-specific adaptations to an extreme high-elevation environment, the Qinghai-Tibet Plateau. Our ancestral trait reconstruction shows that the ancestral snowfinch occupied high elevations and had a larger body mass than most nonsnowfinches in Passeridae. Subsequently, this phenotypic adaptation diversified in the descendant species. By comparing high-quality genomes from representatives of the three phylogenetic lineages, we find that about 95% of genes under positive selection in the descendant species are different from those in the ancestor. Consistently, the biological functions enriched for these species differ from those of their ancestor to various degrees (semantic similarity values ranging from 0.27 to 0.5), suggesting that the three descendant species have evolved divergently from the initial adaptation in their common ancestor. Using a functional assay to a highly selective gene, DTL, we demonstrate that the nonsynonymous substitutions in the ancestor and descendant species have improved the repair capacity of ultraviolet-induced DNA damage. The repair kinetics of the DTL gene shows a twofold to fourfold variation across the ancestor and the descendants. Collectively, this study reveals an exceptional case of adaptive evolution to high-elevation environments, an evolutionary process with an initial adaptation in the common ancestor followed by adaptive diversification of the descendant species.

Structural variations between small alarmone hydrolase dimers support different modes of regulation of the stringent response.

The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelHCg) and Leptospira levettii (RelHLl) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated.

Divergent contributions of coding and noncoding sequences to initial high-altitude adaptation in passerine birds endemic to the Qinghai-Tibet Plateau.

Early events in the evolution of an ancestral lineage can shape the adaptive patterns of descendant species, but the evolutionary mechanisms driving initial adaptation from an ancestor remain largely unexplored. High-altitude adaptations have been extensively explored from the viewpoint of protein-coding genes; however, the contribution of noncoding regions remains relatively neglected. Here, we integrate genomic and transcriptomic data to investigate adaptive evolution in the ancestor of three high-altitude snowfinch species endemic to the Qinghai-Tibet Plateau. Our genome-wide scan for adaptation in the snowfinch ancestor identifies strong adaptation signals in functions of development and metabolism for the coding genes, but in functions of the nervous system development for noncoding regions. This pattern is exclusive to the snowfinch ancestor compared to a control ancestral lineage subject to weak selection. Changes in noncoding regions in the snowfinch ancestor, especially those nearest to coding genes, may be disproportionately associated with the differential expression of genes in the brain tissue compared to other tissues. Extensive gene expression in the brain tissue can be further altered via genetic regulatory networks of transcription factors harbouring potential accelerated regulatory regions (e.g., the development-related transcription factor YEATS4). Altogether, our study provides new evidence concerning how coding and noncoding sequences work through decoupled pathways in initial adaptation to the selective pressure of high-altitude environments. The analysis highlights the idea that noncoding sequences may be promising elements in facilitating the rapid evolution and adaptation to high altitudes.

GenTree, an integrated resource for analyzing the evolution and function of primate-specific coding genes.

The origination of new genes contributes to phenotypic evolution in humans. Two major challenges in the study of new genes are the inference of gene ages and annotation of their protein-coding potential. To tackle these challenges, we created GenTree, an integrated online database that compiles age inferences from three major methods together with functional genomic data for new genes. Genome-wide comparison of the age inference methods revealed that the synteny-based pipeline (SBP) is most suited for recently duplicated genes, whereas the protein-family-based methods are useful for ancient genes. For SBP-dated primate-specific protein-coding genes (PSGs), we performed manual evaluation based on published PSG lists and showed that SBP generated a conservative data set of PSGs by masking less reliable syntenic regions. After assessing the coding potential based on evolutionary constraint and peptide evidence from proteomic data, we curated a list of 254 PSGs with different levels of protein evidence. This list also includes 41 candidate misannotated pseudogenes that encode primate-specific short proteins. Coexpression analysis showed that PSGs are preferentially recruited into organs with rapidly evolving pathways such as spermatogenesis, immune response, mother-fetus interaction, and brain development. For brain development, primate-specific KRAB zinc-finger proteins (KZNFs) are specifically up-regulated in the mid-fetal stage, which may have contributed to the evolution of this critical stage. Altogether, hundreds of PSGs are either recruited to processes under strong selection pressure or to processes supporting an evolving novel organ.

Adaptation and Phenotypic Diversification in Arabidopsis through Loss-of-Function Mutations in Protein-Coding Genes.

According to the less-is-more hypothesis, gene loss is an engine for evolutionary change. Loss-of-function (LoF) mutations resulting in the natural knockout of protein-coding genes not only provide information about gene function but also play important roles in adaptation and phenotypic diversification. Although the less-is-more hypothesis was proposed two decades ago, it remains to be explored on a large scale. In this study, we identified 60,819 LoF variants in 1071 Arabidopsis (Arabidopsis thaliana) genomes and found that 34% of Arabidopsis protein-coding genes annotated in the Columbia-0 genome do not have any LoF variants. We found that nucleotide diversity, transposable element density, and gene family size are strongly correlated with the presence of LoF variants. Intriguingly, 0.9% of LoF variants with minor allele frequency larger than 0.5% are associated with climate change. In addition, in the Yangtze River basin population, 1% of genes with LoF mutations were under positive selection, providing important insights into the contribution of LoF mutations to adaptation. In particular, our results demonstrate that LoF mutations shape diverse phenotypic traits. Overall, our results highlight the importance of the LoF variants for the adaptation and phenotypic diversification of plants.

(p)ppGpp-Dependent Regulation of the Nucleotide Hydrolase PpnN Confers Complement Resistance in Salmonella enterica Serovar Typhimurium.

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria, including Salmonella enterica serovar Typhimurium. S Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues, resulting in severe clinical outcomes. During infection, S Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5'-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane, known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

microRNA-21: a key modulator in oncogenic viral infections.

Oncogenic viruses are associated with approximately 15% of human cancers. In viral infections, microRNAs play an important role in host-pathogen interactions. miR-21 is a highly conserved non-coding RNA that not only regulates the development of oncogenic viral diseases, but also responds to the regulation of intracellular signal pathways. Oncogenic viruses, including HBV, HCV, HPV, and EBV, co-evolve with their hosts and cause persistent infections. The upregulation of host miR-21 manipulates key cellular pathways to evade host immune responses and then promote viral replication. Thus, a better understanding of the role of miR-21 in viral infections may help us to develop effective genetically-engineered oncolytic virus-based therapies against cancer.

LTR-mediated retroposition as a mechanism of RNA-based duplication in metazoans.

In a broad range of taxa, genes can duplicate through an RNA intermediate in a process mediated by retrotransposons (retroposition). In mammals, L1 retrotransposons drive retroposition, but the elements responsible for retroposition in other animals have yet to be identified. Here, we examined young retrocopies from various animals that still retain the sequence features indicative of the underlying retroposition mechanism. In Drosophila melanogaster, we identified and de novo assembled 15 polymorphic retrocopies and found that all retroposed loci are chimeras of internal retrocopies flanked by discontinuous LTR retrotransposons. At the fusion points between the mRNAs and the LTR retrotransposons, we identified shared short similar sequences that suggest the involvement of microsimilarity-dependent template switches. By expanding our approach to mosquito, zebrafish, chicken, and mammals, we identified in all these species recently originated retrocopies with a similar chimeric structure and shared microsimilarities at the fusion points. We also identified several retrocopies that combine the sequences of two or more parental genes, demonstrating LTR-retroposition as a novel mechanism of exon shuffling. Finally, we found that LTR-mediated retrocopies are immediately cotranscribed with their flanking LTR retrotransposons. Transcriptional profiling coupled with sequence analyses revealed that the sense-strand transcription of the retrocopies often lead to the origination of in-frame proteins relative to the parental genes. Overall, our data show that LTR-mediated retroposition is highly conserved across a wide range of animal taxa; combined with previous work from plants and yeast, it represents an ancient and ongoing mechanism continuously shaping gene content evolution in eukaryotes.

Direct measurement of pervasive weak repression by microRNAs and their role at the network level.

BACKGROUND: A gene regulatory network (GRN) comprises many weak links that are often regulated by microRNAs. Since miRNAs rarely repress their target genes by more than 30%, doubts have been expressed about the biological relevance of such weak effects. These doubts raise the possibility of under-estimation as miRNA repression is usually estimated indirectly from equilibrium expression levels. RESULTS: To measure miRNA repression directly, we inhibited transcript synthesis in Drosophila larvae and collected time-course data on mRNA abundance, the decline of which reflects transcript degradation. The rate of target degradation in the absence of miR310s, a moderately expressed miRNA family, was found to decrease by 5 to 15%. A conventional analysis that does not remove transcript synthesis yields an estimate of 6.5%, within the range of the new estimates. These data permit further examinations of the repression mechanisms by miRNAs including seed matching types, APA (alternative polyadenylation) sites, effects of other highly-expressed miRNAs and the length of 3'UTR. Our direct measurements suggest the latter two factors have a measurable effect on decay rate. CONCLUSION: The direct measurement confirms pervasive weak repression by miRNAs, supporting the conclusions based on indirect assays. The confirmation suggests that this weak repression may indeed be miRNAs' main function. In this context, we discuss the recent proposal that weak repression is "cumulatively powerful" in stabilizing GRNs.

Emergence, Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.

A long-term demasculinization of X-linked intergenic noncoding RNAs in Drosophila melanogaster.

Recent studies have revealed key roles of noncoding RNAs in sex-related pathways, but little is known about the evolutionary forces acting on these noncoding RNAs. Profiling the transcriptome of Drosophila melanogaster with whole-genome tiling arrays found that 15% of male-biased transcribed fragments are intergenic noncoding RNAs (incRNAs), suggesting a potentially important role for incRNAs in sex-related biological processes. Statistical analysis revealed a paucity of male-biased incRNAs and coding genes on the X chromosome, suggesting that similar evolutionary forces could be affecting the genomic organization of both coding and noncoding genes. Expression profiling across germline and somatic tissues further suggested that both male meiotic sex chromosome inactivation (MSCI) and sexual antagonism could contribute to the chromosomal distribution of male-biased incRNAs. Comparative sequence analysis showed that the evolutionary age of male-biased incRNAs is a significant predictor of their chromosomal locations. In addition to identifying abundant sex-biased incRNAs in the fly genome, our work unveils a global picture of the complex interplay between noncoding RNAs and sexual chromosome evolution.

Evolution of beak morphology in the Ground Tit revealed by comparative transcriptomics.

BACKGROUND: Beak morphology exhibits considerable adaptive plasticity in birds, which results in highly varied or specialized forms in response to variations in ecology and life history. As the only parid species endemic to the Qinghai-Tibet Plateau, the Ground Tit (Parus humilis) has evolved a distinctly long and curved beak from other parids. An integration of morphometrics, phylogenetics, transcriptomics and embryology allows us to address the evolutionary and developmental mechanisms of the adaptive beak structure observed in the Ground Tit. RESULTS: A morphometric approach quantified that the Ground Tit has a comparatively longer and more decurved upper beaks than other parids. We estimated that the ancestor of the Ground Tit likely had a short straight upper beak similar to most current recognized parid species using an ancestral state reconstruction. This morphological specialization is considered an adaptation to its ground-oriented behavior on the high plateau. To identify genetic mechanisms behind this adaptive change, a comparative transcriptomic analysis was applied between the Ground Tit and its closely related species, the Great Tit (Parus major). We detected that 623 genes were significantly differentially expressed in embryonic upper beaks between the two species, 17 of which were functionally annotated to correlate with bone development and morphogenesis, although genes related to bone development were not found to undergo accelerated evolution in the Ground Tit. RT-qPCR validation confirmed differential expression of five out of eight genes that were selected from the 17 genes. Subsequent functional assays in chicken embryos demonstrated that two of these genes, FGF13 and ITGB3, may affect beak morphology by modulating levels of osteoblasts and osteoclasts. CONCLUSIONS: Our results provide preliminary evidence that development of the long decurved beak of the Ground Tit is likely regulated by transcriptional activities of multiple genes coordinating osteoblasts and osteoclasts. The integration of multiple approaches employed here sheds light on ecological and genetic mechanisms in the evolution of avian morphology.