RESUMO
SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/virologia , Imunidade Inata/genética , Pandemias , SARS-CoV-2/genéticaRESUMO
ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes ß-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , beta-Arrestinas , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Bicamadas Lipídicas , Receptores Acoplados a Proteínas G/metabolismo , Simulação de Dinâmica MolecularRESUMO
The cellular interior is composed of a variety of microenvironments defined by distinct local compositions and composition-dependent intermolecular interactions. We review the various types of nonspecific interactions between proteins and between proteins and other macromolecules and supramolecular structures that influence the state of association and functional properties of a given protein existing within a particular microenvironment at a particular point in time. The present state of knowledge is summarized, and suggestions for fruitful directions of research are offered.
Assuntos
Bioquímica , Proteínas , Substâncias Macromoleculares , Proteínas/química , Proteínas/genéticaRESUMO
Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteoma/genética , Biologia Computacional/métodos , Células HCT116/metabolismo , Células HEK293/metabolismo , Humanos , Espectrometria de Massas/métodos , Mapas de Interação de Proteínas/fisiologia , Proteoma/metabolismo , Proteômica/métodosRESUMO
Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.
Assuntos
Especificidade de Órgãos , Mapeamento de Interação de Proteínas , Animais , Marcação por Isótopo , Masculino , Mamíferos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/química , Movimento Celular , Receptor DCC/deficiência , Receptor DCC/genética , Proteínas Ligadas por GPI/química , Cones de Crescimento/fisiologia , Humanos , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
We have known for decades that long noncoding RNAs (lncRNAs) can play essential functions across most forms of life. The maintenance of chromosome length requires an lncRNA (e.g., hTERC) and two lncRNAs in the ribosome that are required for protein synthesis. Thus, lncRNAs can represent powerful RNA machines. More recently, it has become clear that mammalian genomes encode thousands more lncRNAs. Thus, we raise the question: Which, if any, of these lncRNAs could also represent RNA-based machines? Here we synthesize studies that are beginning to address this question by investigating fundamental properties of lncRNA genes, revealing new insights into the RNA structure-function relationship, determining cis- and trans-acting lncRNAs in vivo, and generating new developments in high-throughput screening used to identify functional lncRNAs. Overall, these findings provide a context toward understanding the molecular grammar underlying lncRNA biology.
Assuntos
Genoma , Biossíntese de Proteínas , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA/genética , Telomerase/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Telomerase/metabolismo , Homeostase do Telômero , Transcrição GênicaRESUMO
Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.
Assuntos
Arrestina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Arrestina/química , Simulação por Computador , Células HEK293 , Humanos , Fosfatos/metabolismo , Fosfopeptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Análise EspectralRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.
Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Splicing de RNA , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , COVID-19/virologia , Chlorocebus aethiops , Células HEK293 , Humanos , Interferons/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , RNA Citoplasmático Pequeno/química , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.
Assuntos
Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas/fisiologia , Espectrometria de Massas/métodos , Plantas/genética , Plantas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodosRESUMO
While knowledge of protein-protein interactions (PPIs) is critical for understanding virus-host relationships, limitations on the scalability of high-throughput methods have hampered their identification beyond a number of well-studied viruses. Here, we implement an in silico computational framework (pathogen host interactome prediction using structure similarity [P-HIPSTer]) that employs structural information to predict â¼282,000 pan viral-human PPIs with an experimental validation rate of â¼76%. In addition to rediscovering known biology, P-HIPSTer has yielded a series of new findings: the discovery of shared and unique machinery employed across human-infecting viruses, a likely role for ZIKV-ESR1 interactions in modulating viral replication, the identification of PPIs that discriminate between human papilloma viruses (HPVs) with high and low oncogenic potential, and a structure-enabled history of evolutionary selective pressure imposed on the human proteome. Further, P-HIPSTer enables discovery of previously unappreciated cellular circuits that act on human-infecting viruses and provides insight into experimentally intractable viruses.
Assuntos
Interações Hospedeiro-Patógeno , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Proteínas Virais/metabolismo , Zika virus/fisiologia , Animais , Atlas como Assunto , Chlorocebus aethiops , Simulação por Computador , Conjuntos de Dados como Assunto , Células HEK293 , Humanos , Células MCF-7 , Proteoma/química , Células Vero , Proteínas Virais/químicaRESUMO
We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.
Assuntos
Ferro/metabolismo , Metaloproteínas/metabolismo , Metformina/farmacologia , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Teste de Complementação Genética , Humanos , Metaloproteínas/genética , Saccharomyces cerevisiae/genéticaRESUMO
Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.
Assuntos
Proteínas de Transporte , Proteínas de Ligação a DNA , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/metabolismo , Fatores de Transcrição , Proteínas Virais , Replicação Viral/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/patologia , Humanos , Mapeamento de Interação de Proteínas , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.
Assuntos
Metaboloma , Proteoma/metabolismo , Proteômica/métodos , Transdução de Sinais , Software , Regulação Alostérica , Sítios de Ligação , Escherichia coli , Metabolômica/métodos , Ligação Proteica , Mapas de Interação de Proteínas , Proteoma/química , Saccharomyces cerevisiae , Análise de Sequência de Proteína/métodosRESUMO
Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Proteínas do Citoesqueleto/química , Humanos , Camundongos , Domínios Proteicos , Splicing de RNA , Alinhamento de Sequência , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Nuclear pore complexes (NPCs) are â¼100 MDa transport channels assembled from multiple copies of â¼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture.
Assuntos
Poro Nuclear/química , Saccharomyces cerevisiae/citologia , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Biogênese de Organelas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/imunologia , Bacteriófagos/genética , Bacteriófagos/imunologia , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/ultraestrutura , DNA Viral/química , Modelos Químicos , Modelos Moleculares , Complexos Multiproteicos/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestruturaRESUMO
Integrator is a multi-subunit protein complex responsible for premature transcription termination of coding and non-coding RNAs. This is achieved via two enzymatic activities, RNA endonuclease and protein phosphatase, acting on the promoter-proximally paused RNA polymerase â ¡ (RNAPâ ¡). Yet, it remains unclear how Integrator assembly and recruitment are regulated and what the functions of many of its core subunits are. Here, we report the structures of two human Integrator sub-complexes: INTS10/13/14/15 and INTS5/8/10/15, and an integrative model of the fully assembled Integrator bound to the RNAPâ ¡ paused elongating complex (PEC). An in silico protein-protein interaction screen of over 1,500 human transcription factors (TFs) identified ZNF655 as a direct interacting partner of INTS13 within the fully assembled Integrator. We propose a model wherein INTS13 acts as a platform for the recruitment of TFs that could modulate the stability of the Integrator's association at specific loci and regulate transcription attenuation of the target genes.
Assuntos
Ligação Proteica , RNA Polimerase II , Fatores de Transcrição , Humanos , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Modelos Moleculares , Microscopia Crioeletrônica , Regiões Promotoras Genéticas , Células HEK293 , Sítios de Ligação , EndorribonucleasesRESUMO
The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.
Assuntos
Proteínas Argonautas , Conformação de Ácido Nucleico , RNA Guia de Sistemas CRISPR-Cas , Complexo de Inativação Induzido por RNA , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/química , Humanos , Complexo de Inativação Induzido por RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/química , Cinética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Interferência de RNA , Sequência de Bases , Células HEK293RESUMO
RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.