Gene Expression Machinery Research Articles

Mechanism of ligand binding signal transduction in a transcriptional riboswitch.

Riboswitches are noncoding RNA molecules that regulate gene expression in bacteria. The riboswitch's aptamer domain (AD) senses the cognate ligand and induces a structural change in the expression platform (EP) to signal on/off to the gene expression machinery. Despite extensive studies, the question of how the ligand binding signal is transmitted from the AD to EP resulting in the structural transitions in the EP remains unanswered. We studied the ligand binding signal transduction from AD to EP in the preQ1-sensing transcriptional riboswitch. In the absence of preQ1, both AD and EP individually form stem-loops with no structural overlap between the two stem-loops. The segment between the AD and EP stem loops is rich in adenine. In the presence of preQ1, AD forms a pseudoknot to encapsulate preQ1. We performed coarse-grained molecular dynamics simulations of full-length preQ1 riboswitch in the presence of Mg2+ and preQ1 ligand. In the absence of preQ1 and variation in Mg2+ concentration ([Mg2+]), we observed that AD populates only stem-loop and does not form the pseudoknot required to capture the ligand. Only in the presence of preQ1 AD attains the pseudoknot conformation by wrapping the A-rich region around the pre-existing stem-loop to facilitate ligand binding. The pseudoknot formation at AD allows EP to form the terminator helix and sequester the poly-U through strand invasion. The wrapping of the A-rich region around the AD stem-loop forces the stem-loop at EP to rupture and slide by 10 nucleotides towards 3'-end and form the terminator helix. The propensity of pseudoknot and terminator formation through strand invasion in the presence of preQ1 is enhanced with increment in [Mg2+]. We show that the competition between base-pair formation and helix sliding rate is crucial for the ligand binding signal transduction.

SLAMseq resolves the kinetics of maternal and zygotic gene expression during early zebrafish embryogenesis.

The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts prevail prior to MZT, we uncover the spurious transcription of hundreds of short and intron-poor genes as early as the 2-cell stage. Upon ZGA, most zygotic transcripts originate from thousands of maternal-zygotic (MZ) genes that are transcribed at rates comparable to those of hundreds of purely zygotic genes and replenish maternal mRNAs at distinct timescales. Rapid replacement of MZ transcripts involves transcript decay features unrelated to major maternal degradation pathways and promotes de novo synthesis of the core gene expression machinery by increasing poly(A)-tail length and translation efficiency. SLAMseq hence provides insights into the timescales, molecular features, and regulation of MZT during zebrafish embryogenesis.

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σB factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σB is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

Principles of gene regulation quantitatively connect DNA to RNA and proteins in bacteria.

Protein concentrations are set by a complex interplay between gene-specific regulatory processes and systemic factors, including cell volume and shared gene expression machineries. Elucidating this interplay is crucial for discerning and designing gene regulatory systems. We quantitatively characterized gene-specific and systemic factors that affect transcription and translation genome-wide for Escherichia coli across many conditions. The results revealed two design principles that make regulation of gene expression insulated from concentrations of shared machineries: RNA polymerase activity is fine-tuned to match translational output, and translational characteristics are similar across most messenger RNAs (mRNAs). Consequently, in bacteria, protein concentration is set primarily at the promoter level. A simple mathematical formula relates promoter activities and protein concentrations across growth conditions, enabling quantitative inference of gene regulation from omics data.

Vacuolal and Peroxisomal Calcium Ion Transporters in Yeasts and Fungi: Key Role in the Translocation of Intermediates in the Biosynthesis of Fungal Metabolites.

Highlights The intracellular calcium content plays a key role in the expression of genes involved in the biosynthesis and secretion of fungal metabolites.The cytosolic calcium concentration in fungi is maintained by influx through the cell membrane and by release from store organelles.Some MSF transporters, e.g., PenV of Penicillium chrysogenum and CefP of Acremonium chrysogenum belong to the TRP calcium ion channels.A few of the numerous calcium ion transporters existing in organelles of different filamentous fungi have been characterized at the functional and subcellular localization levels.The cytosolic calcium signal seems to be transduced by the calcitonin/calcineurin cascade controlling the expression of many fungal genes. The intracellular calcium content in fungal cells is influenced by a large number of environmental and nutritional factors. Sharp changes in the cytosolic calcium level act as signals that are decoded by the cell gene expression machinery, resulting in several physiological responses, including differentiation and secondary metabolites biosynthesis. Expression of the three penicillin biosynthetic genes is regulated by calcium ions, but there is still little information on the role of this ion in the translocation of penicillin intermediates between different subcellular compartments. Using advanced information on the transport of calcium in organelles in yeast as a model, this article reviews the recent progress on the transport of calcium in vacuoles and peroxisomes and its relation to the translocation of biosynthetic intermediates in filamentous fungi. The Penicillium chrysogenum PenV vacuole transporter and the Acremonium chrysogenum CefP peroxisomal transporter belong to the transient receptor potential (TRP) class CSC of calcium ion channels. The PenV transporter plays an important role in providing precursors for the biosynthesis of the tripeptide δ-(-α-aminoadipyl-L-cysteinyl-D-valine), the first intermediate of penicillin biosynthesis in P. chrysogenum. Similarly, CefP exerts a key function in the conversion of isopenicillin N to penicillin N in peroxisomes of A. chrysogenum. These TRP transporters are different from other TRP ion channels of Giberella zeae that belong to the Yvc1 class of yeast TRPs. Recent advances in filamentous fungi indicate that the cytosolic calcium concentration signal is connected to the calcitonin/calcineurin signal transduction cascade that controls the expression of genes involved in the subcellular translocation of intermediates during fungal metabolite biosynthesis. These advances open new possibilities to enhance the expression of important biosynthetic genes in fungi.

Microbiomes, Epigenomics, Immune Response, and Splicing Signatures Interplay: Potential Use of Combination of Regulatory Pathways as Targets for Malignant Mesothelioma.

Malignant mesotheliomas (MM) are hard to treat malignancies with poor prognosis and high mortality rates. This cancer is highly misdiagnosed in Sub-Saharan African countries. According to literature, the incidence of MM is likely to increase particularly in low-middle-income countries (LMICs). The burden of asbestos-induced diseases was estimated to be about 231,000 per annum. Lack of awareness and implementation of regulatory frameworks to control exposure to asbestos fibers contributes to the expected increase. Exposure to asbestos fibers can lead to cancer initiation by several mechanisms. Asbestos-induced epigenetic modifications of gene expression machinery and non-coding RNAs promote cancer initiation and progression. Furthermore, microbiome–epigenetic interactions control the innate and adaptive immunity causing exacerbation of cancer progression and therapeutic resistance. This review discusses epigenetic mechanisms with more focus on miRNAs and their interaction with the microbiome. The potential use of epigenetic alterations and microbiota as specific biomarkers to aid in the early detection and/or development of therapeutic targets is explored. The advancement of combinatorial therapies to prolong overall patient survival or possible eradication of MM especially if it is detected early is discussed.

Genomic sequences and RNA-binding proteins predict RNA splicing efficiency in various single-cell contexts.

The RNA splicing efficiency is of high interest for both understanding the regulatory machinery of gene expression and estimating the RNA velocity in single cells. However, its genomic regulation and stochasticity across contexts remain poorly understood. Here, by leveraging the recent RNA velocity tool, we estimated the relative splicing efficiency across a variety of single-cell RNA-Seq data sets. We further extracted large sets of genomic features and 120 RNA-binding protein features and found they are highly predictive to relative RNA splicing efficiency across multiple tissues and organs on human and mouse. This predictive power brings promise to reveal the complexity of RNA processing and to enhance the analysis of single-cell transcription activities. In order to ensure reproducibility, all preprocessed datasets and scripts used for the prediction and figure generation are publicly available at https://doi.org/10.5281/zenodo.6513669. Supplementary data are available at Bioinformatics online.

Cellular Localization of Dbp2 and Identification of its Interaction Partners

Dbp2 is a DEAD‐box RNA helicase in S. cerevisiae, whose main function is connected to the ribosomal RNA processing and transcription. Interestingly, the localization of Dbp2 is also highly dependent on the presence of glucose, which enables us to study how cellular stress impacts gene expression machinery. Studies from the Tran laboratory show that the rapid nuclear export of Dbp2 in the absence of glucose. Furthermore, unpublished studies from the Tran lab have linked specific phosphorylation events on Dbp2 to glucose‐dependent localization. To determine if phosphorylation affects Dbp2’s function and localization, our first step is to generate mutant Dbp2 proteins that localize to the nucleus or the cytoplasm, irrespective of the presence of glucose. To this end, site‐directed mutagenesis was used on plasmids containing wild type DBP2 genes to convert codons encoding select serine residues to codons that would encode amino acids mimicking the dephosphorylated and phosphorylated state of Dbp2. Following transformation of these mutant‐expressing plasmids and a wild type DBP2 as a control into a dbp2∆strain, a serial dilution spot assay was performed on glucose and galactose plates (mimicking – glucose). We observed that both the phosphorylation mutants (both the non‐phosphorylatable and the phosphor‐mimetic) grew substantially better than our wild type Dbp2 and empty vector. This was unexpected and will be followed up with future studies in the Tran laboratory. In addition to mutant Dbp2 generation, we also set out to identify protein interaction partners of Dbp2 in the cytoplasm. Our goal was to use proximity‐dependent biotin identification (BioID) using an AirID biotin ligase tag to identify Dbp2’s cytoplasmic interaction partners. Following unsuccessful isolation of proteins, immunofluorescence was performed to verify the localization of the ligase tag. Immunofluorescence indicates the AirID tag is more cytoplasmically localized in comparison to nuclear Dbp2, thus, the study was inconclusive. Due to the mislocalization of the AirID tag, in future studies we plan to use different versions of the biotin ligase tag such as the TurboID tag.

Trypanosoma cruzi dysregulates expression of piRNA that can regulate IL‐6 signaling in human cardiac fibroblasts during the early phase of infection

Trypanosoma cruzi, the etiological agent of Chagas Disease, causes severe morbidity, mortality, and economic burden in endemic zones. Due to globalization, the disease is now present in all industrialized regions of the world. About 40% of infected individuals will develop cardiovascular, neurological, and/or gastrointestinal pathologies. Cardiomyopathies induced by chronic parasite infection include organ enlargement and fibrosis, accompanied by significant changes in the cellular expression of profibrotic factors and inflammatory molecules. Accumulating evidence suggests that the parasite induces alterations in host gene expression profiles to facilitate infection and pathogenesis. The role of regulatory gene expression machinery during T. cruzi infection remains to be elucidated. In this study, we aim to evaluate the expression of a class of small non‐coding RNA molecules, piwi‐interacting RNAs (piRNAs), during the early phase of T. cruzi infection in primary human cardiac fibroblasts (PHCF). Recent studies defined a role for STAT3 signaling in the onset of cardiac fibrosis in a Chagasic mouse model. Our bioinformatic analysis of parasite challenged PHCF identified significant modulation of piRNAs targeting IL‐6 signaling pathway components. To validate this, we performed qPCR of IL‐6, SOCS3, and piRNAs predicted to target IL‐6 and SOCS3. Furthermore, we evaluated protein expression for pSTAT3 (Y705) and SOCS3 in cell lysates and IL6 secretion in condition media of parasite challenged PHCF. We observed that the piRNAs expression negatively correlated with their target mRNA and protein expression of IL‐6. This suggests T. cruzi dysregulates the expression of piRNA that can regulate IL‐6 signaling during T. cruzi pathogenesis.

Rewriting the Central Dogma with Synthetic Genetic Polymers.

DNA and RNA are ubiquitous molecules responsible for storage and transmission of genetic information and together comprise the central dogma of molecular biology. However, the recent emergence of synthetic genetic polymers is providing an opportunity to challenge the fundamental principles of life. Herein, we describe the ongoing attempts to rewrite the central dogma with 4'-thioDNA and 4'-thioRNA, which feature a sulfur instead of an oxygen atom in the furanose ring moiety. Using reconstituted Escherichia coli gene expression machinery, studies have shown that the genetic information conserved in 4'-thioDNA can be transcribed to 4'-thioRNA and eventually translated into protein, mirroring the processes that occur in nature. Such studies underscore the feasibility of controlling life by substances other than DNA and RNA.

Neighbours matter: Effects of genomic organization on gene expression plasticity in response to environmental stresses during biological invasions

Gene expression regulation has been widely recognized as an important molecular mechanism underlying phenotypic plasticity in environmental adaptation. However, it remains largely unexplored on the effects of genomic organization on gene expression plasticity under environmental stresses during biological invasions. Here, we use an invasive model ascidian, Ciona robusta, to investigate how genomic organization affects gene expression in response to salinity stresses during range expansions. Our study showed that neighboring genes were co-expressed and approximately 30% of stress responsive genes were physically clustered on chromosomes. Such coordinated expression was substantially affected by the physical distance and orientation of genes. Interestingly, the overall expression correlation of neighboring genes was significantly decreased under high salinity stresses, illustrating that the co-expression regulation could be disrupted by salinity challenges. Furthermore, the clustering of genes was associated with their function constraints and expression patterns - operon genes enriched in gene expression machinery had the highest transcriptional activity and expression stability. Notably, our analyses showed that the tail-to-tail genes, mainly involved in biological functions related to phosphorylation, homeostatic process, and ion transport, exhibited higher intrinsic expression variability and greater response to salinity challenges. Altogether, the results obtained here provide new insights into the effects of gene organization on gene expression plasticity under environmental challenges, hence improving our knowledge on mechanisms of rapid environmental adaptation during biological invasions.

Types and Functions of Mitoribosome-Specific Ribosomal Proteins across Eukaryotes.

Mitochondria are key organelles that combine features inherited from their bacterial endosymbiotic ancestor with traits that arose during eukaryote evolution. These energy producing organelles have retained a genome and fully functional gene expression machineries including specific ribosomes. Recent advances in cryo-electron microscopy have enabled the characterization of a fast-growing number of the low abundant membrane-bound mitochondrial ribosomes. Surprisingly, mitoribosomes were found to be extremely diverse both in terms of structure and composition. Still, all of them drastically increased their number of ribosomal proteins. Interestingly, among the more than 130 novel ribosomal proteins identified to date in mitochondria, most of them are composed of a-helices. Many of them belong to the nuclear encoded super family of helical repeat proteins. Here we review the diversity of functions and the mode of action held by the novel mitoribosome proteins and discuss why these proteins that share similar helical folds were independently recruited by mitoribosomes during evolution in independent eukaryote clades.

Exploring New Functional Aspects of HTLV-1 RNA-Binding Protein Rex: How Does Rex Control Viral Replication?

After integration to the human genome as a provirus, human T-cell leukemia virus type 1 (HTLV-1) utilizes host T cell gene expression machinery for viral replication. The viral RNA-binding protein, Rex, is known to transport unspliced/incompletely spliced viral mRNAs encoding viral structural proteins out of the nucleus to enhance virus particle formation. However, the detailed mechanism of how Rex avoids extra splicing of unspliced/incompletely spliced viral mRNAs and stabilizes them for effective translation is still unclear. To elucidate the underlying molecular mechanism of Rex function, we comprehensively analyzed the changes in gene expression and splicing patterns in Rex-overexpressing T cells. In addition, we identified 81 human proteins interacting with Rex, involved in transcription, splicing, translation, and mRNA quality control. In particular, Rex interacts with NONO and SFPQ, which play important roles in the regulation of transcription and splicing. Accordingly, expression profiles and splicing patterns of a wide variety of genes are significantly changed in Rex-expressing T cells. Especially, the level of vPD-L1 mRNA that lacks the part of exon 4, thus encodes soluble PD-L1 was significantly increased in Rex-expressing cells. Overall, by integrated analysis of these three datasets, we showed for the first time that Rex intervenes the host gene expression machinery throughout the pathway, probably to escort viral unstable mRNAs from transcription (start) to translation (end). Upon exerting its function, Rex may alter the expression level and splicing patterns of various genes, thus influencing the phenotype of the host cell.

Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis.

Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation.

Function of Chloroplasts in Plant Stress Responses.

The chloroplast has a central position in oxygenic photosynthesis and primary metabolism. In addition to these functions, the chloroplast has recently emerged as a pivotal regulator of plant responses to abiotic and biotic stress conditions. Chloroplasts have their own independent genomes and gene-expression machinery and synthesize phytohormones and a diverse range of secondary metabolites, a significant portion of which contribute the plant response to adverse conditions. Furthermore, chloroplasts communicate with the nucleus through retrograde signaling, for instance, reactive oxygen signaling. All of the above facilitate the chloroplast’s exquisite flexibility in responding to environmental stresses. In this review, we summarize recent findings on the involvement of chloroplasts in plant regulatory responses to various abiotic and biotic stresses including heat, chilling, salinity, drought, high light environmental stress conditions, and pathogen invasions. This review will enrich the better understanding of interactions between chloroplast and environmental stresses, and will lay the foundation for genetically enhancing plant-stress acclimatization.

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

Heterogeneity is not always a source of noise: Stochastic gene expression in regulatory heterozygote.

Zygosity of diploid genome (i.e., degree to which two parental alleles of a gene have varied genetic sequences) adds another dimension to stochastic gene expression. The allelic imbalance in chromatin accessibility or divergence in regulatory sequences leads to fitness effects but the quantitative aspects thereof are largely left unexplored. We investigate diploid gene expression systems with homozygous (the same) and heterozygous (varied) combination of alleles in cis-regulatory sequences, not in structural gene loci, and characterize the zygosity-associated stochastic fluctuations in protein abundance. An emerging feat of heterozygosity is its counterintuitive capacity for genetic noise control. Especially when the noise is dominantly contributed to by the fluctuations in duty cycle ("reliability") rather than in process speed ("productivity") of gene expression machinery, its interallelic discrepancy acts to reduce the gene expression noise. These findings offer a novel insight into the rich repertoire of balancing selection enriched in the regulatory elements of immune response genes.

DNA methylation atlas and machinery in the developing and regenerating annelid Platynereis dumerilii

BackgroundMethylation of cytosines in DNA (5mC methylation) is a major epigenetic modification that modulates gene expression and constitutes the basis for mechanisms regulating multiple aspects of embryonic development and cell reprogramming in vertebrates. In mammals, 5mC methylation of promoter regions is linked to transcriptional repression. Transcription regulation by 5mC methylation notably involves the nucleosome remodeling and deacetylase complex (NuRD complex) which bridges DNA methylation and histone modifications. However, less is known about regulatory mechanisms involving 5mC methylation and their function in non-vertebrate animals. In this paper, we study 5mC methylation in the marine annelid worm Platynereis dumerilii, an emerging evolutionary and developmental biology model capable of regenerating the posterior part of its body post-amputation.ResultsUsing in silico and experimental approaches, we show that P. dumerilii displays a high level of DNA methylation comparable to that of mammalian somatic cells. 5mC methylation in P. dumerilii is dynamic along the life cycle of the animal and markedly decreases at the transition between larval to post-larval stages. We identify a full repertoire of mainly single-copy genes encoding the machinery associated with 5mC methylation or members of the NuRD complex in P. dumerilii and show that this repertoire is close to the one inferred for the last common ancestor of bilaterians. These genes are dynamically expressed during P. dumerilii development and regeneration. Treatment with the DNA hypomethylating agent Decitabine impairs P. dumerilii larval development and regeneration and has long-term effects on post-regenerative growth.ConclusionsOur data reveal high levels of 5mC methylation in the annelid P. dumerilii, highlighting that this feature is not specific to vertebrates in the bilaterian clade. Analysis of DNA methylation levels and machinery gene expression during development and regeneration, as well as the use of a chemical inhibitor of DNA methylation, suggest an involvement of 5mC methylation in P. dumerilii development and regeneration. We also present data indicating that P. dumerilii constitutes a promising model to study biological roles and mechanisms of DNA methylation in non-vertebrate bilaterians and to provide new knowledge about evolution of the functions of this key epigenetic modification in bilaterian animals.

Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium.

STUDY QUESTIONAre SARS-CoV-2 canonical cell entry machinery, consisting of ACE2, TMPRSS2, NRP1 and LY6E, or alternative potential cell entry machinery, consisting of BSG, ANPEP, CD209, CLEC4G, TMPRSS4, TMPRSS11A, FURIN, CTSB, CTSL and IFITM1, expressed in the human endometrium across the menstrual cycle?SUMMARY ANSWERAnalysis of cell entry factors for SARS-CoV-2 by single-cell RNA-sequencing (scRNAseq) in the preconceptional human endometrium reveals low risk of infection.WHAT IS KNOWN ALREADYGene expression datasets from bulk endometrial tissue show no significant expression of the SARS-CoV-2 receptor ACE2 and TMPRSS2. This is in contrast to reported expression of ACE2 at the single-cell level in the decidua and trophoblast cells at the maternal–fetal interface in early pregnancy, as well as vertical transmission of SARS-CoV-2 during pregnancy.STUDY DESIGN, SIZE, DURATIONThis analysis of SARS-CoV-2 cell entry machinery gene expression was conducted by scRNAseq in 73 181 human endometrial cells isolated from endometrial biopsies obtained from 27 donors across the menstrual cycle.PARTICIPANTS/MATERIALS, SETTING, METHODSScRNAseq examined the expression of genes encoding cell entry machinery for SARS-CoV-2. The raw data were from a previously published dataset.MAIN RESULTS AND THE ROLE OF CHANCEScRNAseq analysis showed no significant expression of ACE2 in stromal or unciliated epithelial cells in any phase of the menstrual cycle. TMPRSS2 was expressed in epithelial cells during the early proliferative and mid-secretory phases. Interestingly, the expression of NRP1 was observed in both stromal and epithelial cells across all phases of the menstrual cycle, and LY6E was highly expressed in stromal cells. In the mid-secretory phase, coexpression of ACE2 and TMPRSS2 was detected in 0.07% of luminal epithelial cells. No cells simultaneously expressed ACE2, NRP1 and TMPRSS2 at the time of embryo implantation. Focusing on non-canonical cell entry machinery, BSG was highly expressed in all cell types across the menstrual cycle and may interact with CTSB or CTSL proteases, but viral infection using this machinery has not yet been confirmed.LARGE SCALE DATAAll raw data in this study can be found at NCBI’s Gene Expression Omnibus (series accession code GSE111976) and Sequence Read Archive (accession code SRP135922).LIMITATIONS, REASONS FOR CAUTIONOur findings at the single-cell level imply low efficiency of SARS-CoV-2 endometrial infection using canonical receptors in a cohort of healthy reproductive-age women; however, infection of endometrial cells can only be assessed in the presence of the virus. All samples were processed for scRNAseq, so no samples are remaining to analyze protein expression or spatial transcriptomics.WIDER IMPLICATIONS OF THE FINDINGSOur results offer a useful resource to guide reproductive decisions when assessing risk of endometrial infection by SARS-CoV-2 during the preconceptional period in asymptomatic COVID-19 carriers.STUDY FUNDING/COMPETING INTEREST(S)This study was jointly supported by the March of Dimes, Chan Zuckerberg Biohub and MINECO/FEDER (SAF-2015-67164-R, to C.S.) (Spanish Government), and the European Union’s Horizon 2020 Framework Programme for Research and Innovation (Grant agreement 874867). W.W. was supported by the Stanford Bio-X Graduate Bowes Fellowship and Chan Zuckerberg Biohub. F.V. was supported by the Miguel Servet Program Type II of ISCIII (CPII18/00020) and the FIS project (PI18/00957). A patent disclosure has been filed for the study with the title ‘Methods for assessing endometrial transformation’ and the global patent number ‘EP 3807648 A2’ under the inventors S.R.Q., C.S., W.W. and F.V. C.S. is the Founder and Head of the Scientific Advisory Board of Igenomix SL. S.R.Q is the Director of Mirvie. I.M. is partially employed by Igenomix SL. B.R. has no interests to declare.