Computational analysis of super-resolved in situ sequencing data reveals genes modified by immune-tumor contact events.

Cancer cells can manipulate immune cells and escape from the immune system response. Quantifying the molecular changes that occur when an immune cell is touching a tumor cell can increase our understanding of the underlying mechanisms. Recently, it became possible to perform such measurements in situ, for example using expansion sequencing, which enabled in situ sequencing of genes with super-resolution. We systematically examined whether individual immune cells from specific cell types express genes differently when in physical proximity to individual tumor cells. First, we demonstrated that a dense mapping of genes in situ can be utilized for the segmentation of cell bodies in 3D, thus improving our ability to detect likely touching cells. Next, we utilized three different computational approaches to detect the molecular changes that are triggered by proximity: differential expression analysis, tree-based machine learning classifiers, and matrix factorization analysis. This systematic analysis revealed tens of genes, in specific cell types, whose expression separates immune cells that are proximal to tumor cells from those that are not proximal, with a significant overlap between the different detection methods. Remarkably, an order of magnitude more genes are trigger by proximity to tumor cells in CD8 T cells compared to CD4 T cells, in line with the ability of CD8 T cells to directly bind Major Histocompatibility Complex (MHC) Class I on tumor cells. Thus, in situ sequencing of an individual biopsy can be used to detect genes likely involved in immune-tumor cell-cell interactions. The data used in this manuscript and the code of the InSituSeg, Machine learning, cNMF and Moran's I methods are publicly available at https://zenodo.org/record/7845775 (DOI: 10.5281/zenodo.7845775).

Correction: It's All in Your Mind: Determining Germ Cell Fate by Neuronal IRE-1 in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1004747.].

Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity.

Introduction: The success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance. Methods: We report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls. Results: In contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients. Discussion: These features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges.

A somatic hypermutation-based machine learning model stratifies individuals with Crohn's disease and controls.

Crohn's disease (CD) is a chronic relapsing-remitting inflammatory disorder of the gastrointestinal tract that is characterized by altered innate and adaptive immune function. Although massively parallel sequencing studies of the T cell receptor repertoire identified oligoclonal expansion of unique clones, much less is known about the B cell receptor (BCR) repertoire in CD. Here, we present a novel BCR repertoire sequencing data set from ileal biopsies from pediatric patients with CD and controls, and identify CD-specific somatic hypermutation (SHM) patterns, revealed by a machine learning (ML) algorithm trained on BCR repertoire sequences. Moreover, ML classification of a different data set from blood samples of adults with CD versus controls identified that V gene usage, clusters, or mutation frequencies yielded excellent results in classifying the disease (F1 > 90%). In summary, we show that an ML algorithm enables the classification of CD based on unique BCR repertoire features with high accuracy.

Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Germline NPM1 mutations lead to altered rRNA 2'-O-methylation and cause dyskeratosis congenita.

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.

Deciphering the "m6A Code" via Antibody-Independent Quantitative Profiling.

N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.

Publisher Correction: m6A modification controls the innate immune response to infection by targeting type I interferons.

In the version of this article initially published, the penultimate sentence of the abstract included a typographical error ('cxgenes'). The correct word is 'genes'. The error has been corrected in the HTML and PDF version of the article.

m6A modification controls the innate immune response to infection by targeting type I interferons.

N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.

Variants in PUS7 Cause Intellectual Disability with Speech Delay, Microcephaly, Short Stature, and Aggressive Behavior.

We describe six persons from three families with three homozygous protein truncating variants in PUS7: c.89_90del (p.Thr30Lysfs∗20), c.1348C>T (p.Arg450∗), and a deletion of the penultimate exon 15. All these individuals have intellectual disability with speech delay, short stature, microcephaly, and aggressive behavior. PUS7 encodes the RNA-independent pseudouridylate synthase 7. Pseudouridylation is the most abundant post-transcriptional modification in RNA, which is primarily thought to stabilize secondary structures of RNA. We show that the disease-related variants lead to abolishment of PUS7 activity on both tRNA and mRNA substrates. Moreover, pus7 knockout in Drosophila melanogaster results in a number of behavioral defects, including increased activity, disorientation, and aggressiveness supporting that neurological defects are caused by PUS7 variants. Our findings demonstrate that RNA pseudouridylation by PUS7 is essential for proper neuronal development and function.

The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.

Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N1-methyladenosine (m1A), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m1A at single-nucleotide resolution. Within the cytosol, m1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m1A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m1A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m1A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m1A levels was adopted as a potential means of post-transcriptional regulation.

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7.

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks.

TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.

Following synthesis, RNA can be modified with over 100 chemically distinct modifications, which can potentially regulate RNA expression post-transcriptionally. Pseudouridine (Ψ) was recently established to be widespread and dynamically regulated on yeast mRNA, but less is known about Ψ presence, regulation, and biogenesis in mammalian mRNA. Here, we sought to characterize the Ψ landscape on mammalian mRNA, to identify the main Ψ-synthases (PUSs) catalyzing Ψ formation, and to understand the factors governing their specificity toward selected targets. We first developed a framework allowing analysis, evaluation, and integration of Ψ mappings, which we applied to >2.5 billion reads from 30 human samples. These maps, complemented with genetic perturbations, allowed us to uncover TRUB1 and PUS7 as the two key PUSs acting on mammalian mRNA and to computationally model the sequence and structural elements governing the specificity of TRUB1, achieving near-perfect prediction of its substrates (AUC = 0.974). We then validated and extended these maps and the inferred specificity of TRUB1 using massively parallel reporter assays in which we monitored Ψ levels at thousands of synthetically designed sequence variants comprising either the sequences surrounding pseudouridylation targets or systematically designed mutants perturbing RNA sequence and structure. Our findings provide an extensive and high-quality characterization of the transcriptome-wide distribution of pseudouridine in human and the factors governing it and provide an important resource for the community, paving the path toward functional and mechanistic dissection of this emerging layer of post-transcriptional regulation.

The FOXO transcription factor DAF-16 bypasses ire-1 requirement to promote endoplasmic reticulum homeostasis.

The unfolded protein response (UPR) allows cells to adjust the capacity of the endoplasmic reticulum (ER) to the load of ER-associated tasks. We show that activation of the Caenorhabditis elegans transcription factor DAF-16 and its human homolog FOXO3 restore secretory protein metabolism when the UPR is dysfunctional.We show that DAF-16 establishes alternative ER-associated degradation systems that degrade misfolded proteins independently of the ER stress sensor ire-1 and the ER-associated E3 ubiquitin ligase complex sel-11/sel-1. This is achieved by enabling autophagy-mediated degradation and by increasing the levels of skr-5, a component of an ER associated ubiquitin ligase complex. These degradation systems can act together with the conserved UPR to improve ER homeostasis and ER stress resistance, beyond wild-type levels. Because there is no sensor in the ER that activates DAF-16 in response to intrinsic ER stress, natural or artificial interventions that activate DAF-16 may be useful therapeutic approaches to maintain ER homeostasis.

It's all in your mind: determining germ cell fate by neuronal IRE-1 in C. elegans.

The C. elegans germline is pluripotent and mitotic, similar to self-renewing mammalian tissues. Apoptosis is triggered as part of the normal oogenesis program, and is increased in response to various stresses. Here, we examined the effect of endoplasmic reticulum (ER) stress on apoptosis in the C. elegans germline. We demonstrate that pharmacological or genetic induction of ER stress enhances germline apoptosis. This process is mediated by the ER stress response sensor IRE-1, but is independent of its canonical downstream target XBP-1. We further demonstrate that ire-1-dependent apoptosis in the germline requires both CEP-1/p53 and the same canonical apoptotic genes as DNA damage-induced germline apoptosis. Strikingly, we find that activation of ire-1, specifically in the ASI neurons, but not in germ cells, is sufficient to induce apoptosis in the germline. This implies that ER stress related germline apoptosis can be determined at the organism level, and is a result of active IRE-1 signaling in neurons. Altogether, our findings uncover ire-1 as a novel cell non-autonomous regulator of germ cell apoptosis, linking ER homeostasis in sensory neurons and germ cell fate.

A new tool in C. elegans reveals changes in secretory protein metabolism in ire-1-deficient animals.

We recently showed that the ire-1/xbp-1 arm of the UPR plays a crucial role in maintaining basic endoplasmic reticulum (ER) functions required for the metabolism of secreted proteins even during unstressed growth conditions. During these studies we realized that although C. elegans is a powerful system to study the genetics of many cellular processes; it lacks effective tools for tracking the metabolism of secreted proteins at the cell and organism levels. Here, we outline how genetic manipulations and expression analysis of a DAF-28::GFP translational fusion transgene can be combined to infer different steps in the life cycle of secretory proteins. We demonstrate how we have used this tool to reveal folding defects, clearance defects, and secretion defects in ire-1 and xbp-1 mutants. We believe that further studies using this tool will deepen the understanding of secretory protein metabolism.

The ire-1 ER stress-response pathway is required for normal secretory-protein metabolism in C. elegans.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress by adjusting the capacity of the ER to the load of ER-associated tasks. The UPR is important for maintaining ER homeostasis under extreme ER stress. UPR genes are important under normal growth conditions as well, but what they are required for under these conditions is less clear. Using C. elegans, we show that the ire-1/xbp-1 arm of the UPR plays a crucial role in maintaining ER plasticity and function also in the absence of external ER stress. We find that during unstressed growth conditions, loss of ire-1 or xbp-1 compromises basic ER functions required for the metabolism of secreted proteins, including translation, folding and secretion. Notably, by compromising ER-associated degradation (ERAD) and phagocytosis, loss of ire-1 hinders the clearance of misfolded proteins from the ER as well as the clearance of proteins that were secreted into the pseudocoleom. Whereas the basal activity of the UPR is beneficial under normal conditions, it accelerates the pathology caused by toxic Aß protein in a C. elegans model of Alzheimer's disease. Taken together, our findings indicate that UPR genes are critical for maintaining secretory protein metabolism under normal growth conditions.