RESUMO
The human body contains thousands of metabolites derived from mammalian cells, the microbiota, food, and medical drugs. Many bioactive metabolites act through the engagement of G-protein-coupled receptors (GPCRs); however, technological limitations constrain current explorations of metabolite-GPCR interactions. Here, we developed a highly multiplexed screening technology called PRESTO-Salsa that enables simultaneous assessment of nearly all conventional GPCRs (>300 receptors) in a single well of a 96-well plate. Using PRESTO-Salsa, we screened 1,041 human-associated metabolites against the GPCRome and uncovered previously unreported endogenous, exogenous, and microbial GPCR agonists. Next, we leveraged PRESTO-Salsa to generate an atlas of microbiome-GPCR interactions across 435 human microbiome strains from multiple body sites, revealing conserved patterns of cross-tissue GPCR engagement and activation of CD97/ADGRE5 by the Porphyromonas gingivalis protease gingipain K. These studies thus establish a highly multiplexed bioactivity screening technology and expose a diverse landscape of human, diet, drug, and microbiota metabolome-GPCRome interactions.
Assuntos
Microbiota , Receptores Acoplados a Proteínas G , Animais , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Metaboloma , Mamíferos/metabolismoRESUMO
Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.
Assuntos
Simulação por Computador , Edição de Genes , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Edição de Genes/métodos , Conhecimento , RNA Guia de Sistemas CRISPR-Cas/química , Especificidade de Órgãos , Conjuntos de Dados como AssuntoRESUMO
Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.
Assuntos
Proteoma , Proteômica , Proteômica/métodos , Proteoma/metabolismo , Genômica/métodos , Genoma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Microbial culturing and meta-omic profiling technologies have significantly advanced our understanding of the taxonomic and functional variation of the human microbiome and its impact on host processes. The next increase in resolution will come by understanding the role of low-abundant and less-prevalent bacteria and the study of individual cell behaviors that underlie the complexity of microbial ecosystems. To this aim, single-cell techniques are being rapidly developed to isolate, culture, and characterize the genomes and transcriptomes of individual microbes in complex communities. Here, we discuss how these single-cell technologies are providing unique insights into the biology and behavior of human microbiomes.
Assuntos
Microbiota , Bactérias/genética , Genoma Microbiano , Interações entre Hospedeiro e Microrganismos , Humanos , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Bacteria encode reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. We found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when expressed in Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase θ and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ). We also found that a group II intron RT can function similarly in DNA repair, with reciprocal active-site substitutions showing isoleucine favors MMEJ and alanine favors primer extension in both enzymes. These DNA repair functions utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have inherent ability to function in DSBR in a wide range of organisms.
Assuntos
DNA Polimerase Dirigida por RNA , Retroelementos , Alanina/genética , Reparo do DNA por Junção de Extremidades , Reparo do DNA , RNA Polimerases Dirigidas por DNA/genética , Humanos , Íntrons , Isoleucina/genética , DNA Polimerase Dirigida por RNA/químicaRESUMO
Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.
Assuntos
Microscopia , Neurônios , Animais , Interneurônios , Camundongos , Microscopia/métodos , Neurônios/fisiologia , Fótons , VigíliaRESUMO
Comprehensive analysis of neuronal networks requires brain-wide measurement of connectivity, activity, and gene expression. Although high-throughput methods are available for mapping brain-wide activity and transcriptomes, comparable methods for mapping region-to-region connectivity remain slow and expensive because they require averaging across hundreds of brains. Here we describe BRICseq (brain-wide individual animal connectome sequencing), which leverages DNA barcoding and sequencing to map connectivity from single individuals in a few weeks and at low cost. Applying BRICseq to the mouse neocortex, we find that region-to-region connectivity provides a simple bridge relating transcriptome to activity: the spatial expression patterns of a few genes predict region-to-region connectivity, and connectivity predicts activity correlations. We also exploited BRICseq to map the mutant BTBR mouse brain, which lacks a corpus callosum, and recapitulated its known connectopathies. BRICseq allows individual laboratories to compare how age, sex, environment, genetics, and species affect neuronal wiring and to integrate these with functional activity and gene expression.
Assuntos
Conectoma , Regulação da Expressão Gênica , Rede Nervosa/fisiologia , Neurônios/fisiologia , Análise de Sequência de DNA , Animais , Mapeamento Encefálico , Tomada de Decisões , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Reprodutibilidade dos Testes , Análise e Desempenho de TarefasRESUMO
Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Lisossomos/metabolismo , Mitofagia/fisiologia , Animais , Autofagia/fisiologia , Imunofluorescência/métodos , Corantes Fluorescentes/química , Humanos , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitofagia/genéticaRESUMO
Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.
Assuntos
Ensaios de Triagem em Larga Escala , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sistemas CRISPR-Cas/genética , Feminino , Inativação Gênica , Genes Reporter , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células K562 , Lentivirus/fisiologia , Anotação de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição Gênica , Dedos de ZincoRESUMO
The ocean is home to myriad small planktonic organisms that underpin the functioning of marine ecosystems. However, their spatial patterns of diversity and the underlying drivers remain poorly known, precluding projections of their responses to global changes. Here we investigate the latitudinal gradients and global predictors of plankton diversity across archaea, bacteria, eukaryotes, and major virus clades using both molecular and imaging data from Tara Oceans. We show a decline of diversity for most planktonic groups toward the poles, mainly driven by decreasing ocean temperatures. Projections into the future suggest that severe warming of the surface ocean by the end of the 21st century could lead to tropicalization of the diversity of most planktonic groups in temperate and polar regions. These changes may have multiple consequences for marine ecosystem functioning and services and are expected to be particularly significant in key areas for carbon sequestration, fisheries, and marine conservation. VIDEO ABSTRACT.
Assuntos
Biodiversidade , Plâncton/fisiologia , Água do Mar/microbiologia , Geografia , Modelos Teóricos , Oceanos e Mares , FilogeniaRESUMO
Female Aedes aegypti mosquitoes bite humans to obtain blood to develop their eggs. Remarkably, their strong attraction to humans is suppressed for days after the blood meal by an unknown mechanism. We investigated a role for neuropeptide Y (NPY)-related signaling in long-term behavioral suppression and discovered that drugs targeting human NPY receptors modulate mosquito host-seeking. In a screen of all 49 predicted Ae. aegypti peptide receptors, we identified NPY-like receptor 7 (NPYLR7) as the sole target of these drugs. To obtain small-molecule agonists selective for NPYLR7, we performed a high-throughput cell-based assay of 265,211 compounds and isolated six highly selective NPYLR7 agonists that inhibit mosquito attraction to humans. NPYLR7 CRISPR-Cas9 null mutants are defective in behavioral suppression and resistant to these drugs. Finally, we show that these drugs can inhibit biting and blood-feeding on a live host, suggesting a novel approach to control infectious disease transmission by controlling mosquito behavior. VIDEO ABSTRACT.
Assuntos
Comportamento de Busca por Hospedeiro/efeitos dos fármacos , Mosquitos Vetores/efeitos dos fármacos , Receptores de Neuropeptídeo Y/agonistas , Aedes/metabolismo , Animais , Comportamento Alimentar/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Mordeduras e Picadas de Insetos , Receptores de Neuropeptídeo Y/metabolismo , Bibliotecas de Moléculas Pequenas/análiseRESUMO
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
Assuntos
Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , DNA/metabolismo , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Bibliotecas de Moléculas Pequenas , Streptococcus pyogenes/genética , Especificidade por SubstratoRESUMO
Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.
Assuntos
Córtex Auditivo/metabolismo , Rede Nervosa/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Mapeamento Encefálico , Humanos , Integrases/genética , Camundongos , Neuritos/metabolismo , Células Piramidais/metabolismo , Tratos Piramidais/metabolismoRESUMO
Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides because the phenotype, death, selects against itself and has caused a scientific bottleneck that confines research to a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial Display (SLAY), a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened â¼800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences, dramatically increasing the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved. VIDEO ABSTRACT.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Animais , Antibacterianos/química , Escherichia coli , CamundongosRESUMO
A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.
Assuntos
Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Aprendizado Profundo , HumanosRESUMO
Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Secretórias/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/química , Membrana Celular/metabolismo , Microscopia de Fluorescência , Modelos Teóricos , Conformação Proteica , Família de Proteínas da Síndrome de Wiskott-Aldrich/químicaRESUMO
Budding yeasts (subphylum Saccharomycotina) are found in every biome and are as genetically diverse as plants or animals. To understand budding yeast evolution, we analyzed the genomes of 332 yeast species, including 220 newly sequenced ones, which represent nearly one-third of all known budding yeast diversity. Here, we establish a robust genus-level phylogeny comprising 12 major clades, infer the timescale of diversification from the Devonian period to the present, quantify horizontal gene transfer (HGT), and reconstruct the evolution of 45 metabolic traits and the metabolic toolkit of the budding yeast common ancestor (BYCA). We infer that BYCA was metabolically complex and chronicle the tempo and mode of genomic and phenotypic evolution across the subphylum, which is characterized by very low HGT levels and widespread losses of traits and the genes that control them. More generally, our results argue that reductive evolution is a major mode of evolutionary diversification.
Assuntos
Evolução Molecular , Transferência Genética Horizontal , Genoma Fúngico , Filogenia , Saccharomycetales/classificação , Saccharomycetales/genéticaRESUMO
RNAs fold into defined tertiary structures to function in critical biological processes. While quantitative models can predict RNA secondary structure stability, we are still unable to predict the thermodynamic stability of RNA tertiary structure. Here, we probe conformational preferences of diverse RNA two-way junctions to develop a predictive model for the formation of RNA tertiary structure. We quantitatively measured tertiary assembly energetics of >1,000 of RNA junctions inserted in multiple structural scaffolds to generate a "thermodynamic fingerprint" for each junction. Thermodynamic fingerprints enabled comparison of junction conformational preferences, revealing principles for how sequence influences 3-dimensional conformations. Utilizing fingerprints of junctions with known crystal structures, we generated ensembles for related junctions that predicted their thermodynamic effects on assembly formation. This work reveals sequence-structure-energetic relationships in RNA, demonstrates the capacity for diverse compensation strategies within tertiary structures, and provides a path to quantitative modeling of RNA folding energetics based on "ensemble modularity."
Assuntos
RNA/metabolismo , Pareamento Incorreto de Bases , Biblioteca Gênica , Conformação de Ácido Nucleico , Fotodegradação , RNA/química , Dobramento de RNA , Estabilidade de RNA , TermodinâmicaRESUMO
Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.
Assuntos
Técnicas Genéticas , Príons/genética , Engenharia Genética , Técnicas Genéticas/economia , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.