Long-range alternative splicing contributes to neoantigen specificity in glioblastoma.

Recent advances in neoantigen research have accelerated the development of immunotherapies for cancers, such as glioblastoma (GBM). Neoantigens resulting from genomic mutations and dysregulated alternative splicing have been studied in GBM. However, these studies have primarily focused on annotated alternatively-spliced transcripts, leaving non-annotated transcripts largely unexplored. Circular ribonucleic acids (circRNAs), abnormally regulated in tumors, are correlated with the presence of non-annotated linear transcripts with exon skipping events. But the extent to which these linear transcripts truly exist and their functions in cancer immunotherapies remain unknown. Here, we found the ubiquitous co-occurrence of circRNA biogenesis and alternative splicing across various tumor types, resulting in large amounts of long-range alternatively-spliced transcripts (LRs). By comparing tumor and healthy tissues, we identified tumor-specific LRs more abundant in GBM than in normal tissues and other tumor types. This may be attributable to the upregulation of the protein quaking in GBM, which is reported to promote circRNA biogenesis. In total, we identified 1057 specific and recurrent LRs in GBM. Through in silico translation prediction and MS-based immunopeptidome analysis, 16 major histocompatibility complex class I-associated peptides were identified as potential immunotherapy targets in GBM. This study revealed long-range alternatively-spliced transcripts specifically upregulated in GBM may serve as recurrent, immunogenic tumor-specific antigens.

STellaris: a web server for accurate spatial mapping of single cells based on spatial transcriptomics data.

Single-cell RNA sequencing (scRNA-seq) provides insights into gene expression heterogeneities in diverse cell types underlying homeostasis, development and pathological states. However, the loss of spatial information hinders its applications in deciphering spatially related features, such as cell-cell interactions in a spatial context. Here, we present STellaris (https://spatial.rhesusbase.com), a web server aimed to rapidly assign spatial information to scRNA-seq data based on their transcriptomic similarity with public spatial transcriptomics (ST) data. STellaris is founded on 101 manually curated ST datasets comprising 823 sections across different organs, developmental stages and pathological states from humans and mice. STellaris accepts raw count matrix and cell type annotation of scRNA-seq data as the input, and maps single cells to spatial locations in the tissue architecture of properly matched ST section. Spatially resolved information for intercellular communications, such as spatial distance and ligand-receptor interactions (LRIs), are further characterized between annotated cell types. Moreover, we also expanded the application of STellaris in spatial annotation of multiple regulatory levels with single-cell multiomics data, using the transcriptome as a bridge. STellaris was applied to several case studies to showcase its utility of adding value to the ever-growing scRNA-seq data from a spatial perspective.

Direct chemical induction of hepatocyte-like cells with capacity for liver repopulation.

BACKGROUND AND AIMS: Cell fate can be directly reprogrammed from accessible cell types (e.g., fibroblasts) into functional cell types by exposure to small molecule stimuli. However, no chemical reprogramming method has been reported to date that successfully generates functional hepatocyte-like cells that can repopulate liver tissue, casting doubt over the feasibility of chemical reprogramming approaches to obtain desirable cell types for therapeutic applications. APPROACH AND RESULTS: Here, through chemical induction of phenotypic plasticity, we provide a proof-of-concept demonstration of the direct chemical reprogramming of mouse fibroblasts into functional hepatocyte-like cells using exposure to small molecule cocktails in culture medium to successively stimulate endogenous expression of master transcription factors associated with hepatocyte development, such as hepatocyte nuclear factor 4a, nuclear receptor subfamily 1, group I, member 2, and nuclear receptor subfamily 1, group H, member 4. RNA sequencing analysis, metabolic assays, and in vivo physiological experiments show that chemically induced hepatocytes (CiHeps) exhibit comparable activity and function to primary hepatocytes, especially in liver repopulation to rescue liver failure in fumarylacetoacetate hydrolase -/- recombination activating gene 2 -/- interleukin 2 receptor, gamma chain -/- mice in vivo . Single-cell RNA-seq further revealed that gastrointestinal-like and keratinocyte-like cells were induced along with CiHeps, resembling the activation of an intestinal program within hepatic reprogramming as described in transgenic approaches. CONCLUSIONS: Our findings show that direct chemical reprogramming can generate hepatocyte-like cells with high-quality physiological properties, providing a paradigm for establishing hepatocyte identity in fibroblasts and demonstrating the potential for chemical reprogramming in organ/tissue repair and regeneration therapies.

Polyadenylation-related isoform switching in human evolution revealed by full-length transcript structure.

Rhesus macaque is a unique nonhuman primate model for human evolutionary and translational study, but the error-prone gene models critically limit its applications. Here, we de novo defined full-length macaque gene models based on single molecule, long-read transcriptome sequencing in four macaque tissues (frontal cortex, cerebellum, heart and testis). Overall, 8 588 227 poly(A)-bearing complementary DNA reads with a mean length of 14 106 nt were generated to compile the backbone of macaque transcripts, with the fine-scale structures further refined by RNA sequencing and cap analysis gene expression sequencing data. In total, 51 605 macaque gene models were accurately defined, covering 89.7% of macaque or 75.7% of human orthologous genes. Based on the full-length gene models, we performed a human-macaque comparative analysis on polyadenylation (PA) regulation. Using macaque and mouse as outgroup species, we identified 79 distal PA events newly originated in humans and found that the strengthening of the distal PA sites, rather than the weakening of the proximal sites, predominantly contributes to the origination of these human-specific isoforms. Notably, these isoforms are selectively constrained in general and contribute to the temporospatially specific reduction of gene expression, through the tinkering of previously existed mechanisms of nuclear retention and microRNA (miRNA) regulation. Overall, the protocol and resource highlight the application of bioinformatics in integrating multilayer genomics data to provide an intact reference for model animal studies, and the isoform switching detected may constitute a hitherto underestimated regulatory layer in shaping the human-specific transcriptome and phenotypic changes.

A comprehensive RNA editome reveals that edited Azin1 partners with DDX1 to enable hematopoietic stem cell differentiation.

Adenosine-to-inosine RNA editing and the catalyzing enzyme adenosine deaminase are both essential for hematopoietic development and differentiation. However, the RNA editome during hematopoiesis and the underlying mechanisms are poorly defined. Here, we sorted 12 murine adult hematopoietic cell populations at different stages and identified 30 796 editing sites through RNA sequencing. The dynamic landscape of the RNA editome comprises stage- and group-specific and stable editing patterns, but undergoes significant changes during lineage commitment. Notably, we found that antizyme inhibitor 1 (Azin1) was highly edited in hematopoietic stem and progenitor cells (HSPCs). Azin1 editing results in an amino acid change to induce Azin1 protein (AZI) translocation to the nucleus, enhanced AZI binding affinity for DEAD box polypeptide 1 to alter the chromatin distribution of the latter, and altered expression of multiple hematopoietic regulators that ultimately promote HSPC differentiation. Our findings have delineated an essential role for Azin1 RNA editing in hematopoietic cells, and our data set is a valuable resource for studying RNA editing on a more general basis.

ICAM-1-related noncoding RNA accelerates atherosclerosis by amplifying NF-κB signaling.

Long noncoding RNAs (lncRNAs) are critical regulators of inflammation with great potential as new therapeutic targets. However, the role of lncRNAs in early atherosclerosis remains poorly characterized. This study aimed to identify the key lncRNA players in activated endothelial cells (ECs). The lncRNAs in response to pro-inflammatory factors in ECs were screened through RNA sequencing. ICAM-1-related non-coding RNA (ICR) was identified as the most potential candidate for early atherosclerosis. ICR is essential for intercellular adhesion molecule-1 (ICAM1) expression, EC adhesion and migration. In a high fat diet-induced atherosclerosis model in mice, ICR is upregulated in the development of atherosclerosis. After intravenous injection of adenovirus carrying shRNA for mouse ICR, the atherosclerotic plaque area was markedly reduced with the declined expression of ICR and ICAM1. Mechanistically, ICR stabilized the mRNA of ICAM1 in quiescent ECs; while under inflammatory stress, ICR upregulated ICAM1 in a nuclear factor kappa B (NF-κB) dependent manner. RNA-seq analysis showed pro-inflammatory targets of NF-κB were regulated by ICR. Furthermore, the chromatin immunoprecipitation assays showed that p65 binds to ICR promoter and facilitates its transcription. Interestingly, ICR, in turn, promotes p65 accumulation and activity, forming a positive feedback loop to amplify NF-κB signaling. Preventing the degradation of p65 using proteasome inhibitors rescued the expression of NF-κB targets suppressed by ICR. Taken together, ICR acts as an accelerator to amplify NF-κB signaling in activated ECs and suppressing ICR is a promising early intervention for atherosclerosis through ICR/p65 loop blockade.

Diastereoselective and Enantioselective Synthesis of Barbiturate-Fused Spirotetrahydroquinolines via Chiral Palladium(0)/Ligand Complex Catalyzed [4 + 2] Cycloaddition of Vinyl Benzoxazinanones with Barbiturate-Based Olefins.

Under the catalysis of chiral palladium(0)/ligand complex, the [4 + 2] cycloaddition between vinyl benzoxazinanones and barbiturate-based olefins proceeded readily and provided barbiturate-fused spirotetrahydroquinolines in up to 96% chemical yield with up to >99:1 dr and 97% ee. The absolute configuration of barbiturate-fused spirotetrahydroquinolines was clearly identified by X-ray single crystal structure analysis. The reaction mechanism was proposed to shed light on the enantioselective formation of barbiturate-fused spirotetrahydroquinolines.

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 germline-to-soma signal triggers an age-related decline of mitochondrial stress response.

The abilities of an organism to cope with extrinsic stresses and activate cellular stress responses decline during aging. The signals that modulate stress responses in aged animals remain to be elucidated. Here, we discover that feeding Caenorhabditis elegans (C. elegans) embryo lysates to adult worms enabled the animals to activate the mitochondrial unfolded protein response (UPRmt) upon mitochondrial perturbations. This discovery led to subsequent investigations that unveil a hedgehog-like signal that is transmitted from the germline to the soma in adults to inhibit UPRmt in somatic tissues. Additionally, we find that the levels of germline-expressed piRNAs in adult animals markedly decreased. This reduction in piRNA levels coincides with the production and secretion of a hedgehog-like signal and suppression of the UPRmt in somatic cells. Building upon existing research, our study further elucidates the intricate mechanisms of germline-to-soma signaling and its role in modulating the trade-offs between reproduction and somatic maintenance within the context of organismal aging.

Activation of GPR81 by lactate drives tumour-induced cachexia.

Cachexia affects 50-80% of patients with cancer and accounts for 20% of cancer-related death, but the underlying mechanism driving cachexia remains elusive. Here we show that circulating lactate levels positively correlate with the degree of body weight loss in male and female patients suffering from cancer cachexia, as well as in clinically relevant mouse models. Lactate infusion per se is sufficient to trigger a cachectic phenotype in tumour-free mice in a dose-dependent manner. Furthermore, we demonstrate that adipose-specific G-protein-coupled receptor (GPR)81 ablation, similarly to global GPR81 deficiency, ameliorates lactate-induced or tumour-induced adipose and muscle wasting in male mice, revealing adipose GPR81 as the major mediator of the catabolic effects of lactate. Mechanistically, lactate/GPR81-induced cachexia occurs independently of the well-established protein kinase A catabolic pathway, but it is mediated by a signalling cascade sequentially activating Gi-Gßγ-RhoA/ROCK1-p38. These findings highlight the therapeutic potential of targeting GPR81 for the treatment of this life-threatening complication of cancer.

Adaptive functions of structural variants in human brain development.

Quantifying the structural variants (SVs) in nonhuman primates could provide a niche to clarify the genetic backgrounds underlying human-specific traits, but such resource is largely lacking. Here, we report an accurate SV map in a population of 562 rhesus macaques, verified by in-house benchmarks of eight macaque genomes with long-read sequencing and another one with genome assembly. This map indicates stronger selective constrains on inversions at regulatory regions, suggesting a strategy for prioritizing them with the most important functions. Accordingly, we identified 75 human-specific inversions and prioritized them. The top-ranked inversions have substantially shaped the human transcriptome, through their dual effects of reconfiguring the ancestral genomic architecture and introducing regional mutation hotspots at the inverted regions. As a proof of concept, we linked APCDD1, located on one of these inversions and down-regulated specifically in humans, to neuronal maturation and cognitive ability. We thus highlight inversions in shaping the human uniqueness in brain development.

C17orf80 binds the mitochondrial genome to promote its replication.

Serving as the power plant and signaling hub of a cell, mitochondria contain their own genome which encodes proteins essential for energy metabolism and forms DNA-protein assemblies called nucleoids. Mitochondrial DNA (mtDNA) exists in multiple copies within each cell ranging from hundreds to tens of thousands. Maintaining mtDNA homeostasis is vital for healthy cells, and its dysregulation causes multiple human diseases. However, the players involved in regulating mtDNA maintenance are largely unknown though the core components of its replication machinery have been characterized. Here, we identify C17orf80, a functionally uncharacterized protein, as a critical player in maintaining mtDNA homeostasis. C17orf80 primarily localizes to mitochondrial nucleoid foci and exhibits robust double-stranded DNA binding activity throughout the mitochondrial genome, thus constituting a bona fide new mitochondrial nucleoid protein. It controls mtDNA levels by promoting mtDNA replication and plays important roles in mitochondrial metabolism and cell proliferation. Our findings provide a potential target for therapeutics of human diseases associated with defective mtDNA control.

Endothelial Brg1 fine-tunes Notch signaling during zebrafish heart regeneration.

Myocardial Brg1 is essential for heart regeneration in zebrafish, but it remains unknown whether and how endothelial Brg1 plays a role in heart regeneration. Here, we found that both brg1 mRNA and protein were induced in cardiac endothelial cells after ventricular resection and endothelium-specific overexpression of dominant-negative Xenopus Brg1 (dn-xbrg1) inhibited myocardial proliferation and heart regeneration and increased cardiac fibrosis. RNA-seq and ChIP-seq analysis revealed that endothelium-specific overexpression of dn-xbrg1 changed the levels of H3K4me3 modifications in the promoter regions of the zebrafish genome and induced abnormal activation of Notch family genes upon injury. Mechanistically, Brg1 interacted with lysine demethylase 7aa (Kdm7aa) to fine-tune the level of H3K4me3 within the promoter regions of Notch family genes and thus regulated notch gene transcription. Together, this work demonstrates that the Brg1-Kdm7aa-Notch axis in cardiac endothelial cells, including the endocardium, regulates myocardial proliferation and regeneration via modulating the H3K4me3 of the notch promoters in zebrafish.

6mA-Sniper: Quantifying 6mA sites in eukaryotes at single-nucleotide resolution.

While N6-methyldeoxyadenine (6mA) modification is a fundamental regulation in prokaryotes, its prevalence and functions in eukaryotes are controversial. Here, we report 6mA-Sniper to quantify 6mA sites in eukaryotes at single-nucleotide resolution, and delineate a 6mA profile in Caenorhabditis elegans with 2034 sites. Twenty-six of 39 events with Mnl I restriction endonuclease sites were verified, demonstrating the feasibility of this method. The levels of 6mA sites pinpointed by 6mA-Sniper are generally increased after Pseudomonas aeruginosa infection, but decreased in strains with the removal of METL-9, the dominant 6mA methyltransferase. The enrichment of these sites on specific motif of [GC]GAG, the selective constrains on them, and their coordinated changes with METL-9 levels thus support an active shaping of the 6mA profile by methyltransferase. Moreover, for regions marked by 6mA sites that emerged after infection, an enrichment of up-regulated genes was detected, possibly mediated through a mutual exclusive cross-talk between 6mA and H3K27me3 modification. We thus highlight 6mA regulation as a previously neglected regulator in eukaryotes.

A novel N6-Deoxyadenine methyltransferase METL-9 modulates C. elegans immunity via dichotomous mechanisms.

N6-Methyldeoxyadenine (6mA) has been rediscovered as a DNA modification with potential biological function in metazoans. However, the physiological function and regulatory mechanisms regarding the establishment, maintenance and removal of 6mA in eukaryotes are still poorly understood. Here we show that genomic 6mA levels change in response to pathogenic infection in Caenorhabditis elegans (C. elegans). We further identify METL-9 as the methyltransferase that catalyzes DNA 6mA modifications upon pathogen infection. Deficiency of METL-9 impairs the induction of innate immune response genes and renders the animals more susceptible to pathogen infection. Interestingly, METL-9 functions through both 6mA-dependent and -independent mechanisms to transcriptionally regulate innate immunity. Our findings reveal that 6mA is a functional DNA modification in immunomodulation in C. elegans.

De novo genes with an lncRNA origin encode unique human brain developmental functionality.

Human de novo genes can originate from neutral long non-coding RNA (lncRNA) loci and are evolutionarily significant in general, yet how and why this all-or-nothing transition to functionality happens remains unclear. Here, in 74 human/hominoid-specific de novo genes, we identified distinctive U1 elements and RNA splice-related sequences accounting for RNA nuclear export, differentiating mRNAs from lncRNAs, and driving the origin of de novo genes from lncRNA loci. The polymorphic sites facilitating the lncRNA-mRNA conversion through regulating nuclear export are selectively constrained, maintaining a boundary that differentiates mRNAs from lncRNAs. The functional new genes actively passing through it thus showed a mode of pre-adaptive origin, in that they acquire functions along with the achievement of their coding potential. As a proof of concept, we verified the regulations of splicing and U1 recognition on the nuclear export efficiency of one of these genes, the ENSG00000205704, in human neural progenitor cells. Notably, knock-out or over-expression of this gene in human embryonic stem cells accelerates or delays the neuronal maturation of cortical organoids, respectively. The transgenic mice with ectopically expressed ENSG00000205704 showed enlarged brains with cortical expansion. We thus demonstrate the key roles of nuclear export in de novo gene origin. These newly originated genes should reflect the novel uniqueness of human brain development.

TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis.

How amphipathic phospholipids are shuttled between the membrane bilayer remains an essential but elusive process, particularly at the endoplasmic reticulum (ER). One prominent phospholipid shuttling process concerns the biogenesis of APOB-containing lipoproteins within the ER lumen, which may require bulk trans-bilayer movement of phospholipids from the cytoplasmic leaflet of the ER bilayer. Here, we show that TMEM41B, present in the lipoprotein export machinery, encodes a previously conceptualized ER lipid scramblase mediating trans-bilayer shuttling of bulk phospholipids. Loss of hepatic TMEM41B eliminates plasma lipids, due to complete absence of mature lipoproteins within the ER, but paradoxically also activates lipid production. Mechanistically, scramblase deficiency triggers unique ER morphological changes and unsuppressed activation of SREBPs, which potently promotes lipid synthesis despite stalled secretion. Together, this response induces full-blown nonalcoholic hepatosteatosis in the TMEM41B-deficient mice within weeks. Collectively, our data uncovered a fundamental mechanism safe-guarding ER function and integrity, dysfunction of which disrupts lipid homeostasis.

HLH-11 modulates lipid metabolism in response to nutrient availability.

The ability of organisms to sense nutrient availability and tailor their metabolic states to withstand nutrient deficiency is critical for survival. To identify previously unknown regulators that couple nutrient deficiency to body fat utilization, we performed a cherry-picked RNAi screen in C. elegans and found that the transcription factor HLH-11 regulates lipid metabolism in response to food availability. In well-fed worms, HLH-11 suppresses transcription of lipid catabolism genes. Upon fasting, the HLH-11 protein level is reduced through lysosome- and proteasome-mediated degradation, thus alleviating the inhibitory effect of HLH-11, activating the transcription of lipid catabolism genes, and utilizing fat. Additionally, lipid profiling revealed that reduction in the HLH-11 protein level remodels the lipid landscape in C. elegans. Moreover, TFAP4, the mammalian homolog of HLH-11, plays an evolutionarily conserved role in regulating lipid metabolism in response to starvation. Thus, TFAP4 may represent a potential therapeutic target for lipid storage disorders.

NDUFAB1 confers cardio-protection by enhancing mitochondrial bioenergetics through coordination of respiratory complex and supercomplex assembly.

The impairment of mitochondrial bioenergetics, often coupled with exaggerated reactive oxygen species (ROS) production, is a fundamental disease mechanism in organs with a high demand for energy, including the heart. Building a more robust and safer cellular powerhouse holds the promise for protecting these organs in stressful conditions. Here, we demonstrate that NADH:ubiquinone oxidoreductase subunit AB1 (NDUFAB1), also known as mitochondrial acyl carrier protein, acts as a powerful cardio-protector by conferring greater capacity and efficiency of mitochondrial energy metabolism. In particular, NDUFAB1 not only serves as a complex I subunit, but also coordinates the assembly of respiratory complexes I, II, and III, and supercomplexes, through regulating iron-sulfur biosynthesis and complex I subunit stability. Cardiac-specific deletion of Ndufab1 in mice caused defective bioenergetics and elevated ROS levels, leading to progressive dilated cardiomyopathy and eventual heart failure and sudden death. Overexpression of Ndufab1 effectively enhanced mitochondrial bioenergetics while limiting ROS production and protected the heart against ischemia-reperfusion injury. Together, our findings identify that NDUFAB1 is a crucial regulator of mitochondrial energy and ROS metabolism through coordinating the assembly of respiratory complexes and supercomplexes, and thus provide a potential therapeutic target for the prevention and treatment of heart failure.

N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation.

N6-methyldeoxyadenine (6mA), a major type of DNA methylation in bacteria, represents a part of restriction-modification systems to discriminate host genome from invader DNA1. With the recent advent of more sensitive detection techniques, 6mA has also been detected in some eukaryotes2-8. However, the physiological function of this epigenetic mark in eukaryotes remains elusive. Heritable changes in DNA 5mC methylation have been associated with transgenerational inheritance of responses to a high-fat diet9, thus raising the exciting possibility that 6mA may also be transmitted across generations and serve as a carrier of inheritable information. Using Caenorhabditis elegans as a model, here we report that histone H3K4me3 and DNA 6mA modifications are required for the transmission of mitochondrial stress adaptations to progeny. Intriguingly, the global DNA 6mA level is significantly elevated following mitochondrial perturbation. N6-methyldeoxyadenine marks mitochondrial stress response genes and promotes their transcription to alleviate mitochondrial stress in progeny. These findings suggest that 6mA is a precisely regulated epigenetic mark that modulates stress response and signals transgenerational inheritance in C. elegans.