RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to -1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site-a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.
Assuntos
Microscopia Crioeletrônica , Mudança da Fase de Leitura do Gene Ribossômico , Conformação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , SARS-CoV-2/química , SARS-CoV-2/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Humanos , Modelos Moleculares , COVID-19/virologiaRESUMO
Influenza A virus (IAV) is a respiratory virus that causes epidemics and pandemics. Knowledge of IAV RNA secondary structure in vivo is crucial for a better understanding of virus biology. Moreover, it is a fundament for the development of new RNA-targeting antivirals. Chemical RNA mapping using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) coupled with Mutational Profiling (MaP) allows for the thorough examination of secondary structures in low-abundance RNAs in their biological context. So far, the method has been used for analyzing the RNA secondary structures of several viruses including SARS-CoV-2 in virio and in cellulo. Here, we used SHAPE-MaP and dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for genome-wide secondary structure analysis of viral RNA (vRNA) of the pandemic influenza A/California/04/2009 (H1N1) strain in both in virio and in cellulo environments. Experimental data allowed the prediction of the secondary structures of all eight vRNA segments in virio and, for the first time, the structures of vRNA5, 7, and 8 in cellulo. We conducted a comprehensive structural analysis of the proposed vRNA structures to reveal the motifs predicted with the highest accuracy. We also performed a base-pairs conservation analysis of the predicted vRNA structures and revealed many highly conserved vRNA motifs among the IAVs. The structural motifs presented herein are potential candidates for new IAV antiviral strategies.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Humanos , Vírus da Influenza A Subtipo H1N1/genética , SARS-CoV-2/genética , Vírus da Influenza A/genética , RNA Viral/genética , GenômicaRESUMO
RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.
Assuntos
Reagentes de Ligações Cruzadas/química , Vírus da Influenza A/química , Influenza Humana/virologia , RNA Viral/química , Tiouridina/química , Pareamento de Bases , Sequência de Bases , Humanos , Conformação de Ácido NucleicoRESUMO
Influenza A virus (IAV) is a member of the single-stranded RNA (ssRNA) family of viruses. The most recent global pandemic caused by the SARS-CoV-2 virus has shown the major threat that RNA viruses can pose to humanity. In comparison, influenza has an even higher pandemic potential as a result of its high rate of mutations within its relatively short (<13 kbp) genome, as well as its capability to undergo genetic reassortment. In light of this threat, and the fact that RNA structure is connected to a broad range of known biological functions, deeper investigation of viral RNA (vRNA) structures is of high interest. Here, for the first time, we propose a secondary structure for segment 8 vRNA (vRNA8) of A/California/04/2009 (H1N1) formed in the presence of cellular and viral components. This structure shows similarities with prior in vitro experiments. Additionally, we determined the location of several well-defined, conserved structural motifs of vRNA8 within IAV strains with possible functionality. These RNA motifs appear to fold independently of regional nucleoprotein (NP)-binding affinity, but a low or uneven distribution of NP in each motif region is noted. This research also highlights several accessible sites for oligonucleotide tools and small molecules in vRNA8 in a cellular environment that might be a target for influenza A virus inhibition on the RNA level.
Assuntos
Regulação Viral da Expressão Gênica , Genoma Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Conformação de Ácido Nucleico , RNA Viral/química , Animais , Sequência de Bases , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Modelos Moleculares , Motivos de Nucleotídeos/genética , Dobramento de RNA , RNA Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.
Assuntos
Proteínas de Bactérias/química , Ribonucleoproteína Nuclear Heterogênea A1/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Pressão , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
MYC pre-mRNA is spliced with high fidelity to produce the transcription factor known to regulate cellular differentiation, proliferation, apoptosis, and alternative splicing. The mechanisms underpinning the pre-mRNA splicing of MYC, however, remain mostly unexplored. In this study, we examined the interaction of heterogeneous nuclear ribonucleoprotein C (HNRNPC) with MYC intron 2. Building off published eCLIP studies, we confirmed this interaction with poly(U) regions in intron 2 of MYC and found that full binding is correlated with optimal protein production. The interaction appears to be compensatory, as mutational disruption of all three poly(U) regions was required to reduce both HNRNPC binding capacity and fidelity of either splicing or translation. Poly(U) sequences in MYC intron 2 were relatively conserved across sequences from several different species. Lastly, we identified a short sequence just upstream of an HNRNPC binding region that when removed enhances MYC translation.
Assuntos
Precursores de RNA , Splicing de RNA , Íntrons/genética , Precursores de RNA/genética , Processamento Alternativo , MutaçãoRESUMO
SARS-CoV-2 is a betacoronavirus that causes COVID-19, a global pandemic that has resulted in many infections, deaths, and socio-economic challenges. The virus has a large positive-sense, single-stranded RNA genome of â¼30 kb, which produces subgenomic RNAs (sgRNAs) through discontinuous transcription. The most abundant sgRNA is sgRNA N, which encodes the nucleocapsid (N) protein. In this study, we probed the secondary structure of sgRNA N and a shorter model without a 3' UTR in vitro, using the SHAPE (selective 2'-hydroxyl acylation analyzed by a primer extension) method and chemical mapping with dimethyl sulfate and 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate. We revealed the secondary structure of sgRNA N and its shorter variant for the first time and compared them with the genomic RNA N structure. Based on the structural information, we designed gapmers, siRNAs and antisense oligonucleotides (ASOs) to target the N protein coding region of sgRNA N. We also generated eukaryotic expression vectors containing the complete sequence of sgRNA N and used them to screen for new SARS-CoV-2 gene N expression inhibitors. Our study provides novel insights into the structure and function of sgRNA N and potential therapeutic tools against SARS-CoV-2.
Assuntos
Conformação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , Replicação Viral , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Replicação Viral/efeitos dos fármacos , RNA Viral/genética , Humanos , Antivirais/farmacologia , Antivirais/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/antagonistas & inibidores , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Ésteres do Ácido Sulfúrico/farmacologia , Ésteres do Ácido Sulfúrico/química , COVID-19/virologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/química , Genoma Viral , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/químicaRESUMO
Major advances in RNA secondary structural motif prediction have been achieved in the last few years; however, few methods harness the predictive power of multiple approaches to deliver in-depth characterizations of local RNA motifs and their potential functionality. Additionally, most available methods do not predict RNA pseudoknots. This work combines complementary bioinformatic systems into one robust discovery pipeline where: â¢RNA sequences are folded to search for thermodynamically favorable motifs utilizing ScanFold.â¢Motifs are expanded and refolded into alternate pseudoknot conformations by Knotty/Iterative HFold.â¢All conformations are evaluated for covariance via the cm-builder pipeline (Infernal and R-scape).
RESUMO
Influenza virus is a persistent threat to human health; indeed, the deadliest modern pandemic was in 1918 when an H1N1 virus killed an estimated 50 million people globally. The intent of this work is to better understand influenza from an RNA-centric perspective to provide local, structural motifs with likely significance to the influenza infectious cycle for therapeutic targeting. To accomplish this, we analyzed over four hundred thousand RNA sequences spanning three major clades: influenza A, B and C. We scanned influenza segments for local secondary structure, identified/modeled motifs of likely functionality, and coupled the results to an analysis of evolutionary conservation. We discovered 185 significant regions of predicted ordered stability, yet evidence of sequence covariation was limited to 7 motifs, where 3-found in influenza C-had higher than expected amounts of sequence covariation.
Assuntos
Betainfluenzavirus/genética , Gammainfluenzavirus/genética , Vírus da Influenza A/genética , Estabilidade de RNA , RNA Viral/ultraestrutura , Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Betainfluenzavirus/efeitos dos fármacos , Gammainfluenzavirus/efeitos dos fármacos , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Análise de Sequência de RNA , Relação Estrutura-AtividadeRESUMO
SARS-CoV-2 has exploded throughout the human population. To facilitate efforts to gain insights into SARS-CoV-2 biology and to target the virus therapeutically, it is essential to have a roadmap of likely functional regions embedded in its RNA genome. In this report, we used a bioinformatics approach, ScanFold, to deduce the local RNA structural landscape of the SARS-CoV-2 genome with the highest likelihood of being functional. We recapitulate previously-known elements of RNA structure and provide a model for the folding of an essential frameshift signal. Our results find that SARS-CoV-2 is greatly enriched in unusually stable and likely evolutionarily ordered RNA structure, which provides a large reservoir of potential drug targets for RNA-binding small molecules. Results are enhanced via the re-analyses of publicly-available genome-wide biochemical structure probing datasets that are broadly in agreement with our models. Additionally, ScanFold was updated to incorporate experimental data as constraints in the analysis to facilitate comparisons between ScanFold and other RNA modelling approaches. Ultimately, ScanFold was able to identify eight highly structured/conserved motifs in SARS-CoV-2 that agree with experimental data, without explicitly using these data. All results are made available via a public database (the RNAStructuromeDB: https://structurome.bb.iastate.edu/sars-cov-2) and model comparisons are readily viewable at https://structurome.bb.iastate.edu/sars-cov-2-global-model-comparisons.
RESUMO
SARS-CoV-2 is a positive-sense single-stranded RNA virus that has exploded throughout the global human population. This pandemic coronavirus strain has taken scientists and public health researchers by surprise and knowledge of its basic biology (e.g. structure/function relationships in its genomic, messenger and template RNAs) and modes for therapeutic intervention lag behind that of other human pathogens. In this report we used a recently-developed bioinformatics approach, ScanFold, to deduce the RNA structural landscape of the SARS-CoV-2 transcriptome. We recapitulate known elements of RNA structure and provide a model for the folding of an essential frameshift signal. Our results find that the SARS-CoV-2 is greatly enriched in unusually stable and likely evolutionarily ordered RNA structure, which provides a huge reservoir of potential drug targets for RNA-binding small molecules. Our results also predict regions that are accessible for intermolecular interactions, which can aid in the design of antisense therapeutics. All results are made available via a public database (the RNAStructuromeDB) where they may hopefully drive drug discovery efforts to inhibit SARS-CoV-2 pathogenesis.
RESUMO
BACKGROUND: The course of idiopathic pulmonary fibrosis (IPF) is uncertain with variable patterns of disease progression. We sought to evaluate the prognostic utility of the WBC, a routinely performed lab test, in a well-defined cohort of outpatient IPF subjects. METHODS: We reviewed IPF patient records from two independent ILD centers (Inova Fairfax in Falls Church, VA, USA and Ege University Hospital in Izmir, Turkey) between 2007 and 2018. Demographics, CBC data, and patient outcomes were obtained. Survival differences were analyzed. RESULTS: There were 436 IPF outpatients in the cohort with a median WBC of 8.9 × 109 cells per liter. For pragmatic purposes, patients were categorized into two groups, WBC ≥9 or WBC <9. Patients with WBC <9 had a median transplant-free survival of 50.5 months from the time of the CBC, compared to 32.4 months for those with WBC ≥9 (p < 0.0001). The association between WBC and attenuated survival remained significant after adjusting for GAP stage, steroid use, and antifibrotic use when WBC was analyzed both as a continuous (HR: 1.11; 95% CI: 1.05-1.17) and a dichotomized variable (high (WBC ≥9) vs. low (WBC <9), (HR: 1.53; 95% CI:1.09-2.15). WBC and absolute neutrophil count (ANC) were highly correlated suggesting that PMNs account for most of this association (r = 0.92). CONCLUSIONS: Baseline WBC may impart important and readily available prognostic information in outpatients with IPF. Further studies are warranted to validate this as a potential biomarker for IPF, as well as to define the biologic basis for the association.
Assuntos
Fibrose Pulmonar Idiopática/diagnóstico , Contagem de Leucócitos , Idoso , Biomarcadores/sangue , Feminino , Humanos , Fibrose Pulmonar Idiopática/sangue , Masculino , Pessoa de Meia-Idade , Neutrófilos , Pacientes Ambulatoriais , Prognóstico , Taxa de SobrevidaRESUMO
COVID-19 is a global pandemic, thus requiring multiple strategies to develop modalities against it. Herein, we designed multiple bioactive small molecules that target a functional structure within the SARS-CoV-2's RNA genome, the causative agent of COVID-19. An analysis to characterize the structure of the RNA genome provided a revised model of the SARS-CoV-2 frameshifting element, in particular its attenuator hairpin. By studying an RNA-focused small molecule collection, we identified a drug-like small molecule (C5) that avidly binds to the revised attenuator hairpin structure with a K d of 11 nM. The compound stabilizes the hairpin's folded state and impairs frameshifting in cells. The ligand was further elaborated into a ribonuclease targeting chimera (RIBOTAC) to recruit a cellular ribonuclease to destroy the viral genome (C5-RIBOTAC) and into a covalent molecule (C5-Chem-CLIP) that validated direct target engagement and demonstrated its specificity for the viral RNA, as compared to highly expressed host mRNAs. The RIBOTAC lead optimization strategy improved the bioactivity of the compound at least 10-fold. Collectively, these studies demonstrate that the SARS-CoV-2 RNA genome should be considered druggable.