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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.

Motivation The rise in the number of genotyped ancient individuals provides an opportunity to estimate population admixture models for many populations. However, in models describing modern populations as mixtures of ancient ones, it is typically difficult to estimate the model mixing coefficients and to evaluate its fit to the data. Results We present LINADMIX, designed to tackle this problem by solving a constrained linear model when both the ancient and the modern genotypes are represented in a low-dimensional space. LINADMIX estimates the mixing coefficients and their standard errors, and computes a P-value for testing the model fit to the data. We quantified the performance of LINADMIX using an extensive set of simulated studies. We show that LINADMIX can accurately estimate admixture coefficients, and is robust to factors such as population size, genetic drift, proportion of missing data and various types of model misspecification. Availability and implementation LINADMIX is available as a python code at https://github.com/swidler/linadmix. Supplementary information Supplementary data are available at Bioinformatics online.

Background Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene. Methods We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2′-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele. Results A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2′-Methoxy Ethyl modification (2′MOE). Conclusion The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.

Forty years ago, in a seminal paper published in Science, Settle and Patterson used archeological and historical data to estimate the rates of worldwide lead production since the discovery of cupellation, approximately 5000 years ago. Here, we record actual lead exposure of a human population by direct measurements of the concentrations of lead in petrous bones of individuals representing approximately 12 000 years of inhabitation in Italy. This documentation of lead pollution throughout human history indicates that, remarkably, much of the estimated dynamics in lead production is replicated in human exposure. Thus, lead pollution in humans has closely followed anthropogenic lead production. This observation raises concerns that the forecasted increase in the production of lead and other metals might affect human health in the near future.

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.

Recent advances in ancient DNA extraction and high-throughput sequencing technologies enabled the high-quality sequencing of archaic genomes, including the Neanderthal and the Denisovan. While comparisons with modern humans revealed both archaic-specific and human-specific sequence changes, in the absence of gene expression information, understanding the functional implications of such genetic variations remains a major challenge. To study gene regulation in archaic humans, epigenetic research comes to our aid. DNA methylation, which is highly correlated with transcription, can be directly measured in modern samples, as well as reconstructed in ancient samples. This puts DNA methylation as a natural basis for comparative epigenetics between modern humans, archaic humans and nonhuman primates.

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.

In mammals, transposable elements are largely silenced, but under fortuitous circumstances may be co-opted to play a functional role. Here, we show that when Alu elements are inserted within or nearby genes in sense orientation, they may contribute to the transcriptome diversity by forming new cleavage and polyadenylation sites. We mapped these new gene ends in human onto the Alu sequence and identified three hotspots of cleavage and polyadenylation site formation. Interestingly, the native Alu sequence does not contain any canonical polyadenylation signal. We therefore studied what evolutionary processes might explain the formation of these specific hotspots of novel gene ends. We show that two of the three hotspots might have emerged from mutational processes that turned sequences that resemble polyadenylation signals into full-blown canonical signals, whereas one hotspot is tightly linked to the process of Alu insertion into the genome. Overall, Alu elements may lie behind the formation of 302 new gene end variants, affecting a total of 243 genes. Intergenic Alu elements may elongate genes by creating a downstream cleavage site, intronic Alu elements may lead to gene variants which code for truncated proteins, and 3'UTR Alu elements may result in gene variants with alternative 3'UTR.

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.

The 3'-end of the coding sequence in several species is known to show specific codon usage bias. Several factors have been suggested to underlie this phenomenon, including selection against translation efficiency, selection for translation accuracy, and selection against RNA folding. All are supported by some evidence, but there is no general agreement as to which factors are the main determinants. Nor is it known how universal this phenomenon is, and whether the same factors explain it in different species. To answer these questions, we developed a measure that quantifies the codon usage bias at the gene end, and used it to compute this bias for 91 species that span the three domains of life. In addition, we characterized the codons in each species by features that allow discrimination between the different factors. Combining all these data, we were able to show that there is a universal trend to favor AT-rich codons toward the gene end. Moreover, we suggest that this trend is explained by avoidance from forming RNA secondary structures around the stop codon, which may interfere with normal translation termination.

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).

Summary Understanding the ecological function of an animal's pigmentation pattern is an intriguing research challenge. We used quantitative information on lizard foraging behaviour to search for movement correlates of patterns across taxa. We hypothesized that noticeable longitudinal stripes that enhance escape by motion dazzle are advantageous for mobile foragers that are highly detectable against the stationary background. Cryptic pigmentation patterns are beneficial for less-mobile foragers that rely on camouflage to reduce predation. Using an extensive literature survey and phylogenetically controlled analyses, we found that striped lizards were substantially more mobile than lizards with cryptic patterns. The percentage of time spent moving was the major behavioural index responsible for this difference. We provide empirical support for the hypothesized association between lizard dorsal pigmentation patterns and foraging behaviour. Our simple yet comprehensive explanation may be relevant to many other taxa that present variation in body pigmentation patterns.

Recessive CRB2 mutations were recently reported to cause both steroid resistant nephrotic syndrome and prenatal onset ventriculomegaly with kidney disease. We report two Ashkenazi Jewish siblings clinically diagnosed with ciliopathy. Both presented with severe congenital hydrocephalus and mild urinary tract anomalies. One affected sibling also has lung hypoplasia and heart defects. Exome sequencing and further CRB2 analysis revealed that both siblings are compound heterozygotes for CRB2 mutations p.N800K and p.Gly1036Alafs*43, and heterozygous for a deleterious splice variant in the ciliopathy gene TTCB21. CRB2 is a polarity protein which plays a role in ciliogenesis and ciliary function. Biallelic CRB2 mutations in animal models result in phenotypes consistent with ciliopathy. This report expands the phenotype of CRB2 mutations to include lung hypoplasia and uretero-pelvic renal anomalies, and confirms cardiac malformation as a feature. We suggest that CRB2-associated disease is a new ciliopathy syndrome with possible digenic/triallelic inheritance, as observed in other ciliopathies. Clinically, CRB2 should be assessed when ciliopathy is suspected, especially in Ashkenazi Jews, where we found that p.N800K carrier frequency is 1 of 64. Patients harboring CRB2 mutations should be tested for the complete range of ciliopathy manifestations.

We report genome-wide DNA data for 73 individuals from five archaeological sites across the Bronze and Iron Ages Southern Levant. These individuals, who share the ‘‘Canaanite’’ material culture, can be modeled as descending from two sources: (1) earlier local Neolithic populations and (2) populations related to the Chalcolithic Zagros or the Bronze Age Caucasus. The non-local contribution increased over time, as evinced by three outliers who can be modeled as descendants of recent migrants. We show evidence that different ‘‘Canaanite’’ groups genetically resemble each other more than other populations. We find that Levant-related modern populations typically have substantial ancestry coming from populations related to the Chalcolithic Zagros and the Bronze Age Southern Levant. These groups also harbor ancestry from sources we cannot fully model with the available data, highlighting the critical role of post- Bronze-Age migrations into the region over the past 3, 000 years.

MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the expression and translation of genes in healthy and diseased tissues. Herein, we characterize short RNAs from human HeLa cells found in the supraspliceosome, a nuclear dynamic machine in which pre-mRNA processing occurs. We sequenced small RNAs (

Intron positions upon the mRNA transcript are sometimes remarkably conserved even across distantly related eukaryotic species. This has made the comparison of intron-exon architectures across orthologous transcripts a very useful tool for studying various evolutionary processes. Moreover, the wide range of functions associated with introns may confer biological meaning to evolutionary changes in gene architectures. Yet, there is currently no database that offers such comparative information. Here, we present JuncDB (http://juncdb.carmelab.huji.ac.il/), an exon-exon junction database dedicated to the comparison of architectures between orthologous transcripts. It covers nearly 40,000 sets of orthologous transcripts spanning 88 eukaryotic species. JuncDB offers a user-friendly interface, access to detailed information, instructive graphical displays of the comparative data and easy ways to download data to a local computer. In addition, JuncDB allows the analysis to be carried out either on specific genes, or at a genome-wide level for any selected group of species.

BackgroundFamilial glucocorticoid deficiency (FGD) reflects specific failure of adrenocortical glucocorticoid production in response to adrenocorticotropic hormone (ACTH). Most cases are caused by mutations encoding ACTH-receptor components (MC2R, MRAP) or the general steroidogenesis protein (StAR). Recently, nicotinamide nucleotide transhydrogenase (NNT) mutations were found to cause FGD through a postulated mechanism resulting from decreased detoxification of reactive oxygen species (ROS) in adrenocortical cells.Methods and resultsIn a consanguineous Palestinian family with combined mineralocorticoid and glucocorticoid deficiency, whole-exome sequencing revealed a novel homozygous NNT_c.598 G>A, p.G200S, mutation. Another affected, unrelated Palestinian child was also homozygous for NNT_p.G200S. Haplotype analysis showed this mutation is ancestral; carrier frequency in ethnically matched controls is 1/200. Assessment of patient fibroblasts for ROS production, ATP content and mitochondrial morphology showed that biallelic NNT mutations result in increased levels of ROS, lower ATP content and morphological mitochondrial defects.ConclusionsThis report of a novel NNT mutation, p.G200S, expands the phenotype of NNT mutations to include mineralocorticoid deficiency. We provide the first patient-based evidence that NNT mutations can cause oxidative stress and both phenotypic and functional mitochondrial defects. These results directly demonstrate the importance of NNT to mitochondrial function in the setting of adrenocortical insufficiency.

An appreciable fraction of introns is thought to be involved in cellular functions, but there is no obvious way to predict which specific intron is likely to be functional. For each intron we are given a feature representation that is based on its evolutionary patterns. For a small subsets of introns we are also given an indication that they are functional. For all other introns it is not known whether they are functional or not. Our task is to estimate what fraction of introns are functional and, how likely it is that each individual intron is functional. We define a probabilistic classification model that treats the given functionality labels as noisy versions of labels created by a Deep Neural Network model. The maximum-likelihood model parameters are found by utilizing the Expectation-Maximization algorithm. We show that roughly 80% of the functional introns are still not recognized as such, and that roughly a third of all introns are functional.

One of the biggest challenges in studying how genes work is understanding their effect on the physiology and anatomy of the body. Existing tools try to address this using indirect features, such as expression levels and biochemical pathways. Here, we present Gene ORGANizer (geneorganizer.huji.ac.il), a phenotype-based tool that directly links human genes to the body parts they affect. It is built upon an exhaustive curated database that links more than 7,000 genes to ∼150 anatomical parts using >150,000 gene-organ associations. The tool offers user-friendly platforms to analyze the anatomical effects of individual genes, and identify trends within groups of genes. We demonstrate how Gene ORGANizer can be used to make new discoveries, showing that chromosome X is enriched with genes affecting facial features, that positive selection targets genes with more constrained phenotypic effects, and more. We expect Gene ORGANizer to be useful in a variety of evolutionary, medical and molecular studies aimed at understanding the phenotypic effects of genes.

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.