RESUMO
Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Regiões 3' não Traduzidas/genética , RNA de Cadeia Dupla , Doenças Neuroinflamatórias , Inflamação , Receptores de Reconhecimento de Padrão/genética , NeurôniosRESUMO
Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.
Assuntos
Antivirais , Farmacorresistência Viral , SARS-CoV-2 , Humanos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , COVID-19/virologia , Lactamas , Leucina , Nitrilas , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , SARS-CoV-2/crescimento & desenvolvimento , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Mutação , Especificidade por Substrato , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Replicação Viral/efeitos dos fármacos , Desenho de Fármacos , ProlinaRESUMO
Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis; however, its effects on cancer progression remain poorly understood. Here, to address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed dedicated regression-based methods to analyse ribosome profiling and alternative polyadenylation data, and identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as a translational controller of a specific mRNA regulon. We found that HNRNPC is downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3' untranslated region lengthening and, subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. In addition, the reduced expression of HNRNPC and its regulon is associated with the worse prognosis in breast cancer patient cohorts.
Assuntos
Neoplasias da Mama , Processamento Pós-Transcricional do RNA , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Antisense RNAs are ubiquitous in human cells, yet their role is largely unexplored. Here we profiled antisense RNAs in the MDA-MB-231 breast cancer cell line and its highly lung metastatic derivative. We identified one antisense RNA that drives cancer progression by upregulating the redox enzyme NADPH quinone dehydrogenase 1 (NQO1), and named it NQO1-AS. Knockdown of either NQO1 or NQO1-AS reduced lung colonization in a mouse model, and investigation into the role of NQO1 indicated that it is broadly protective against oxidative damage and ferroptosis. Breast cancer cells in the lung are dependent on this pathway, and this dependence can be exploited therapeutically by inducing ferroptosis while inhibiting NQO1. Together, our findings establish a role for NQO1-AS in the progression of breast cancer by regulating its sense mRNA post-transcriptionally. Because breast cancer predominantly affects females, the disease models used in this study are of female origin and the results are primarily applicable to females.
Assuntos
Neoplasias da Mama , Segunda Neoplasia Primária , Neoplasias Cutâneas , Animais , Camundongos , Feminino , Humanos , Neoplasias da Mama/genética , RNA Antissenso , Quinonas/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Melanoma Maligno CutâneoRESUMO
Dysregulated inflammation within the central nervous system (CNS) contributes to neuropathology in infectious, autoimmune, and neurodegenerative disease. With the exception of microglia, major histocompatibility complex (MHC) proteins are virtually undetectable in the mature, healthy central nervous system (CNS). Neurons have generally been considered incapable of antigen presentation, and although interferon gamma (IFN-γ) can elicit neuronal MHC class I (MHC-I) expression and antigen presentation in vitro, it has been unclear whether similar responses occur in vivo. Here we directly injected IFN-γ into the ventral midbrain of mature mice and analyzed gene expression profiles of specific CNS cell types. We found that IFN-γ upregulated MHC-I and associated mRNAs in ventral midbrain microglia, astrocytes, oligodendrocytes, and GABAergic, glutamatergic, and dopaminergic neurons. The core set of IFN-γ-induced genes and their response kinetics were similar in neurons and glia, but with a lower amplitude of expression in neurons. A diverse repertoire of genes was upregulated in glia, particularly microglia, which were the only cells to undergo cellular proliferation and express MHC classII (MHC-II) and associated genes. To determine if neurons respond directly via cell-autonomous IFN-γ receptor (IFNGR) signaling, we produced mutant mice with a deletion of the IFN-γ-binding domain of IFNGR1 in dopaminergic neurons, which resulted in a complete loss of dopaminergic neuronal responses to IFN-γ. Our results demonstrate that IFN-γ induces neuronal IFNGR signaling and upregulation of MHC-I and related genes in vivo, although the expression level is low compared to oligodendrocytes, astrocytes, and microglia.
Assuntos
Interferon gama , Doenças Neurodegenerativas , Camundongos , Animais , Interferon gama/metabolismo , Doenças Neurodegenerativas/metabolismo , Sistema Nervoso Central/metabolismo , Astrócitos/metabolismo , Mesencéfalo/metabolismoRESUMO
Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-19 (refs. 1,2). However, because SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here we examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both and their sequences showed a multitude of 3CL protease mutations. In the experiment peformed with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Nevertheless, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations mediated only low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (around 100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next-generation protease inhibitors.
Assuntos
Antivirais , COVID-19 , Farmacorresistência Viral , SARS-CoV-2 , Humanos , Antivirais/farmacologia , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Mutação , Tratamento Farmacológico da COVID-19RESUMO
The SARS-CoV-2 3CL protease (3CLpro) is an attractive therapeutic target, as it is essential to the virus and highly conserved among coronaviruses. However, our current understanding of its tolerance to mutations is limited. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the 3CLpro and validate a subset of our results within authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein. Yet, we also identify specific residues that appear immutable, suggesting that these may be targets for future 3CLpro inhibitors. Finally, we utilize our screening as a basis to identify E166V as a resistance-conferring mutation against the clinically used 3CLpro inhibitor, nirmatrelvir. Collectively, the functional map presented herein may serve as a guide to better understand the biological properties of the 3CLpro and for drug development against coronaviruses.
Assuntos
COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Peptídeo Hidrolases/genética , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , SARS-CoV-2/genéticaRESUMO
Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-19 1,2 . However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities 3â"9 , there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro , and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.
RESUMO
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CL pro ), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CL pro , and validate our results both in yeast and in authentic viruses. We reveal that the 3CL pro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CL pro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CL pro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.
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Aberrant alternative splicing is a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. Here, we report a systematic effort to decipher the RNA structural code that shapes pathological splicing during breast cancer metastasis. We discovered a previously unknown structural splicing enhancer that is enriched near cassette exons with increased inclusion in highly metastatic cells. We show that the spliceosomal protein small nuclear ribonucleoprotein polypeptide A' (SNRPA1) interacts with these enhancers to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion, in part through SNRPA1-mediated regulation of PLEC alternative splicing, which can be counteracted by splicing modulating morpholinos. Our findings establish a noncanonical regulatory role for SNRPA1 as a prometastatic splicing enhancer in breast cancer.
Assuntos
Processamento Alternativo , Neoplasias da Mama/patologia , Metástase Neoplásica/genética , RNA/genética , RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Algoritmos , Animais , Sítios de Ligação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Progressão da Doença , Éxons , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Conformação de Ácido Nucleico , Plectina/genética , Ligação Proteica , Interferência de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , RNA-Seq , Ribonucleoproteína Nuclear Pequena U2/genética , Software , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Identifying master regulators that drive pathologic gene expression is a key challenge in precision oncology. Here, we have developed an analytic framework, named PRADA, that identifies oncogenic RNA-binding proteins through the systematic detection of coordinated changes in their target regulons. Application of this approach to data collected from clinical samples, patient-derived xenografts, and cell line models of colon cancer metastasis revealed the RNA-binding protein RBMS1 as a suppressor of colon cancer progression. We observed that silencing RBMS1 results in increased metastatic capacity in xenograft mouse models, and that restoring its expression blunts metastatic liver colonization. We have found that RBMS1 functions as a posttranscriptional regulator of RNA stability by directly binding its target mRNAs. Together, our findings establish a role for RBMS1 as a previously unknown regulator of RNA stability and as a suppressor of colon cancer metastasis with clinical utility for risk stratification of patients. SIGNIFICANCE: By applying a new analytic approach to transcriptomic data from clinical samples and models of colon cancer progression, we have identified RBMS1 as a suppressor of metastasis and as a post-transcriptional regulator of RNA stability. Notably, RBMS1 silencing and downregulation of its targets are negatively associated with patient survival.See related commentary by Carter, p. 1261.This article is highlighted in the In This Issue feature, p. 1241.
Assuntos
Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Colo/patologia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Mucosa Intestinal/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Estadiamento de Neoplasias , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , RNA-Seq , Regulon , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.
Assuntos
Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Here we performed a systematic search to identify breast-cancer-specific small noncoding RNAs, which we have collectively termed orphan noncoding RNAs (oncRNAs). We subsequently discovered that one of these oncRNAs, which originates from the 3' end of TERC, acts as a regulator of gene expression and is a robust promoter of breast cancer metastasis. This oncRNA, which we have named T3p, exerts its prometastatic effects by acting as an inhibitor of RISC complex activity and increasing the expression of the prometastatic genes NUPR1 and PANX2. Furthermore, we have shown that oncRNAs are present in cancer-cell-derived extracellular vesicles, raising the possibility that these circulating oncRNAs may also have a role in non-cell autonomous disease pathogenesis. Additionally, these circulating oncRNAs present a novel avenue for cancer fingerprinting using liquid biopsies.
Assuntos
Neoplasias da Mama/genética , Ácidos Nucleicos Livres/genética , MicroRNAs/genética , Pequeno RNA não Traduzido/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Carboxipeptidases/genética , Proliferação de Células/genética , Ácidos Nucleicos Livres/sangue , Conexinas/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , MicroRNAs/classificação , Metástase Neoplásica , Proteínas de Neoplasias/genética , Pequeno RNA não Traduzido/sangueRESUMO
We present a new upsampling method to enhance the spatial resolution of depth images. Given a low-resolution depth image from an active depth sensor and a potentially high-resolution color image from a passive RGB camera, we formulate it as an adaptive cost aggregation problem and solve it using the bilateral filter. The formulation synergistically combines the median and bilateral filters thus it better preserves the depth edges and is more robust to noise. Numerical and visual evaluations on a total of 37 Middlebury data sets demonstrate the effectiveness of our method. A real-time high-resolution depth capturing system is also developed using commercial active depth sensor based on the proposed upsampling method.
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This paper presents a new approach for stereo matching and view interpolation problems based on triangular tessellations suitable for a linear array of rectified cameras. The domain of the reference image is initially partitioned into triangular regions using edge and scale information, aiming to place vertices along image edges and increase the number of triangles in textured regions. A region-based matching algorithm is then used to find an initial disparity for each triangle, and a refinement stage is applied to change the disparity at the vertices of the triangles, generating a piecewise linear disparity map. A simple post-processing procedure is applied to connect triangles with similar disparities generating a full 3D mesh related to each camera (view), which are used to generate new synthesized views along the linear camera array. With the proposed framework, view interpolation reduces to the trivial task of rendering polygonal meshes, which can be done very fast, particularly when GPUs are employed. Furthermore, the generated views are hole-free, unlike most point-based view interpolation schemes that require some kind of post-processing procedures to fill holes.