RESUMO
Sialylation, the addition of sialic acid to glycans, is a crucial post-translational modification of proteins, contributing to neurodevelopment, oncogenesis, and immune response. In cancer, sialylation is dramatically upregulated. Yet, the functional biochemical consequences of sialylation remain mysterious. Here, we establish a µMap proximity labeling platform that utilizes metabolically inserted azidosialic acid to introduce iridium-based photocatalysts on sialylated cell-surface glycoproteins as a means to profile local microenvironments across the sialylated proteome. In comparative experiments between primary cervical cells and a cancerous cell line (HeLa), we identify key differences in both the global sialome and proximal proteins, including solute carrier proteins that regulate metabolite and ion transport. In particular, we show that cell-surface interactions between receptors trafficking ethanolamine and zinc are sialylation-dependent and impact intracellular metabolite levels. These results establish a µMap method for interrogating proteoglycan function and support a role for sialylated glycoproteins in regulating cell-surface transporters.
Assuntos
Glicoproteínas , Ácido N-Acetilneuramínico , Humanos , Glicoproteínas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Glicoproteínas de Membrana/metabolismo , Membrana Celular/metabolismo , Transporte de Íons , Polissacarídeos/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of the COVID-19 pandemic, remains a global medical problem. Angiotensin-converting enzyme 2 (ACE2) was identified as the primary viral entry receptor, and transmembrane serine protease 2 primes the spike protein for membrane fusion. However, ACE2 expression is generally low and variable across tissues, suggesting that auxiliary receptors facilitate viral entry. Identifying these factors is critical for understanding SARS-Cov-2 pathophysiology and developing new countermeasures. However, profiling host-virus interactomes involves extensive genetic screening or complex computational predictions. Here, we leverage the photocatalytic proximity labeling platform µMap to rapidly profile the spike interactome in human cells and identify eight novel candidate receptors. We systemically validate their functionality in SARS-CoV-2 pseudoviral uptake assays with both Wuhan and Delta spike variants and show that dual expression of ACE2 with either neuropilin-2, ephrin receptor A7, solute carrier family 6 member 15, or myelin and lymphocyte protein 2 significantly enhances viral uptake. Collectively, our data show that SARS-CoV-2 synergistically engages several host factors for cell entry and establishes µMap as a powerful tool for rapidly interrogating host-virus interactomes.
Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do VírusRESUMO
Modern proximity labeling techniques have enabled significant advances in understanding biomolecular interactions. However, current tools primarily utilize activation modes that are incompatible with complex biological environments, limiting our ability to interrogate cell- and tissue-level microenvironments in animal models. Here, we report µMap-Red, a proximity labeling platform that uses a red-light-excited SnIV chlorin e6 catalyst to activate a phenyl azide biotin probe. We validate µMap-Red by demonstrating photonically controlled protein labeling in vitro through several layers of tissue, and we then apply our platform in cellulo to label EGFR microenvironments and validate performance with STED microscopy and quantitative proteomics. Finally, to demonstrate labeling in a complex biological sample, we deploy µMap-Red in whole mouse blood to profile erythrocyte cell-surface proteins. This work represents a significant methodological advance toward light-based proximity labeling in complex tissue environments and animal models.
Assuntos
Biotina , Proteômica , Animais , Biotina/metabolismo , Luz , Proteínas de Membrana , Camundongos , Proteômica/métodos , Coloração e RotulagemRESUMO
Adenosine-to-inosine (A-to-I) editing is a conserved eukaryotic RNA modification that contributes to development, immune response, and overall cellular function. Here, we utilize Endonucleaseâ V (EndoV), which binds specifically to inosine in RNA, to develop an EndoV-linked immunosorbency assay (EndoVLISA) as a rapid, plate-based chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimize and validate our assay with chemically synthesized oligonucleotides. We then demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current standard RNA-seq approaches. Lastly, we deploy our EndoVLISA for profiling differential A-to-I RNA editing signatures in normal and diseased human tissue, illustrating the utility of our platform as a diagnostic bioassay. Together, the EndoVLISA method is cost-effective, straightforward, and utilizes common laboratory equipment, offering a highly accessible new approach for studying A-to-I editing. Moreover, the multi-well plate format makes this the first assay amenable for direct high-throughput quantification of A-to-I editing for applications in disease detection and drug development.
Assuntos
Adenosina/química , Inosina/química , Medições Luminescentes , RNA/análise , Humanos , Edição de RNARESUMO
Creating accurate maps of A-to-I RNA editing activity is vital to improving our understanding of the biological role of this process and harnessing it as a signal for disease diagnosis. Current RNA sequencing techniques are susceptible to random sampling limitations due to the complexity of the transcriptome and require large amounts of RNA material, specialized instrumentation, and high read counts to accurately interrogate A-to-I editing sites. To address these challenges, we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA. While Mg2+ enables eEndoV to catalyze RNA cleavage, we show that similar levels of Ca2+ instead promote binding of inosine without cleavage and thus enable high affinity capture of inosine in RNA. We leverage this capability to demonstrate EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing) as a facile and effective method to enrich A-to-I edited transcripts prior to RNA-seq, producing significant increases in the coverage and detection of identified editing sites. We envision the use of this approach as a straightforward and cost-effective strategy to improve the epitranscriptomic informational density of RNA samples, facilitating a deeper understanding of the functional roles of A-to-I editing.
Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/química , Inosina/química , Edição de RNA , RNA Mensageiro/química , Cálcio/química , Escherichia coli/enzimologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de RNA/métodosRESUMO
Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.
Assuntos
Glioxal/química , Ácidos Nucleicos/química , Sequência de Bases , Catálise , Domínio Catalítico , Metilação , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Relação Estrutura-Atividade , Biologia Sintética , Tetroses/química , TermodinâmicaRESUMO
Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.
Assuntos
Acrilamida/química , Adenosina/análise , Química Click , Inosina/análise , Edição de RNA , RNA/química , RNA/metabolismo , Adenosina/metabolismo , Inosina/metabolismoRESUMO
Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive cross-linker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.
Assuntos
Corantes Fluorescentes/química , Marcadores de Fotoafinidade/química , Proteínas/química , Animais , Humanos , Microscopia de Fluorescência , Corantes de Rosanilina/químicaRESUMO
Adenosine-to-inosine (A-to-I) RNA editing is a widespread and conserved post-transcriptional modification, producing significant changes in cellular function and behavior. Accurately identifying, detecting, and quantifying these sites in the transcriptome is necessary to improve our understanding of editing dynamics, its broader biological roles, and connections with diseases. Chemical labeling of edited bases coupled with affinity enrichment has enabled improved characterization of several forms of RNA editing. However, there are no approaches currently available for pull-down of inosines. To address this need, we explore acrylamide as a labeling motif and report here an acrylamidofluorescein reagent that reacts with inosine and enables enrichment of inosine-containing RNA transcripts. This method provides improved sensitivity in the detection and identification of inosines toward a more comprehensive transcriptome-wide analysis of A-to-I editing. Acrylamide derivatization is also highly generalizable, providing potential for the labeling of inosine with a wide variety of probes and affinity handles.
Assuntos
Marcadores de Afinidade/química , Fluoresceína/química , Inosina/química , RNA/química , Sequenciamento de Nucleotídeos em Larga Escala , Edição de RNARESUMO
Adenosine-to-inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions, and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed endonuclease V (EndoV), a magnesium-dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces an endonuclease V immunostaining assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.
RESUMO
Adenosine-to-Inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed Endonuclease V (EndoV), a magnesium dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces Endonuclease V Immunostaining Assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.
RESUMO
Understanding the intricate network of biomolecular interactions that govern cellular processes is a fundamental pursuit in biology. Over the past decade, photocatalytic proximity labeling has emerged as one of the most powerful and versatile techniques for studying these interactions as well as uncovering subcellular trafficking patterns, drug mechanisms of action, and basic cellular physiology. In this article, we review the basic principles, methodologies, and applications of photocatalytic proximity labeling as well as examine its modern development into currently available platforms. We also discuss recent key studies that have successfully leveraged these technologies and importantly highlight current challenges faced by the field. Together, this review seeks to underscore the potential of photocatalysis in proximity labeling for enhancing our understanding of cell biology while also providing perspective on technological advances needed for future discovery.
Assuntos
Processos Fotoquímicos , Catálise , Humanos , Coloração e Rotulagem/métodos , AnimaisRESUMO
The deamination of adenosine to inosine is an important modification in nucleic acids that functionally recodes the identity of the nucleobase to a guanosine. Current methods to analyze and detect this single nucleotide change, such as sequencing and PCR, typically require time-consuming or costly procedures. Alternatively, fluorescent "turn-on" probes that result in signal enhancement in the presence of target are useful tools for real-time detection and monitoring of nucleic acid modification. Here we describe forced-intercalation PNA (FIT-PNA) probes that are designed to bind to inosine-containing nucleic acids and use thiazole orange (TO), 4-dimethylamino-naphthalimide (4DMN), and malachite green (MG) fluorogenic dyes to detect A-to-I editing events. We show that incorporation of the dye as a surrogate base negatively affects the duplex stability but does not abolish binding to targets. We then determined that the identity of the adjacent nucleobase and temperature affect the overall signal and fluorescence enhancement in the presence of inosine, achieving an 11-fold increase, with a limit of detection (LOD) of 30 pM. We determine that TO and 4DMN probes are viable candidates to enable selective inosine detection for biological applications.
RESUMO
RNA undergoes extensive biochemical modification following transcription. In addition to RNA splicing, transcripts are processed by a suite of enzymes that alter the chemical structure of different nucleobases. Broadly termed as 'RNA editing,' these modifications impart significant functional changes to translation, localization, and stability of individual transcripts within the cell. These changes are dynamic and required for a number of critical cellular processes, and dysregulation of these pathways is responsible for several disease states. Accurately detecting, measuring, and mapping different RNA modifications across the transcriptome is vital to understanding their broader functions as well as leveraging these events as diagnostic biomarkers. Here, we review recent advances in profiling several types of RNA modifications, with particular emphasis on adenosine-to-inosine (A-to-I) and N6-methyladenosine (m6A) RNA editing. We especially highlight approaches that utilize proteins to detect or enrich modified RNA transcripts before sequencing, and we summarize recent insights yielded from these techniques.
Assuntos
Inosina , Edição de RNA , Adenosina/metabolismo , Inosina/metabolismo , RNA/genética , RNA/metabolismo , TranscriptomaRESUMO
Adenosine to-inosine (A-to-I) RNA editing is a conserved post-transcriptional modification that is critical for a variety of cellular processes. A-to-I editing is widespread in nearly all types of RNA, directly imparting significant global changes in cellular function and behavior. Dysfunctional RNA editing is also implicated in a number of diseases, and A-to-I editing activity is rapidly becoming an important biomarker for early detection of cancer, immune disorders, and neurodegeneration. While millions of sites have been identified, the biological function of the majority of these sites is unknown, and the regulatory mechanisms for controlling editing activity at individual sites is not well understood. Robust detection and mapping of A-to-I editing activity throughout the transcriptome is vital for understanding these properties and how editing affects cellular behavior. However, accurately identifying A-to-I editing sites is challenging because of inherent sampling errors present in RNA-seq. We recently developed Endonuclease V immunoprecipitation enrichment sequencing (EndoVIPER-seq) to directly address this challenge by enrichment of A-to-I edited RNAs prior to sequencing. This protocol outlines how to process cellular RNA, enrich for A-to-I edited transcripts with EndoVIPER pulldown, and prepare libraries suitable for generating RNA-seq data. © 2020 Wiley Periodicals LLC. Basic Protocol 1: mRNA fragmentation and glyoxalation Basic Protocol 2: EndoVIPER pulldown Basic Protocol 3: RNA-seq library preparation and data analysis.
Assuntos
Adenosina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Inosina/genética , Edição de RNA/genética , RNA/genética , Adenosina/metabolismo , Inosina/metabolismo , RNA/metabolismoRESUMO
Fluorescence microscopy has dramatically advanced our understanding of the processes that drive biological systems by enabling the imaging and tracking of biomolecules of interest inside of living cells. In particular, proteins of interest can be genetically tagged with fluorescent proteins or labeled with small molecule fluorophore probes to enable visualization. However, both of these methods are generally limited in signal-to-background resolution and options are limited for achieving temporal control over labeling. Photoreactive "fluorogenic" dyes can overcome these limitations and enable user-defined crosslinking with low background fluorescence. In this chapter, we discuss current approaches for live cell protein labeling with particular emphasis on the novel use of photoreactive fluorogenic dyes for protein imaging. We further describe in detail the synthesis and characterization of a fluorogenic malachite green probe functionalized with a photoreactive diazirine crosslinker and illustrate how to apply this probe toward covalent photoaffinity labeling and imaging of target proteins in live cells.