RESUMEN
The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/ß-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems.
Asunto(s)
Citoesqueleto , Eritrocitos , Animales , Humanos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Eritrocitos/citología , Eritrocitos/metabolismo , Espectrina/análisis , Espectrina/metabolismo , PorcinosRESUMEN
Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.
Asunto(s)
ARN , Retroelementos , ARN/metabolismo , División del ADN , ADN Polimerasa Dirigida por ARN/metabolismo , Transcripción ReversaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding of the RNA virus and its interactions with host proteins could improve therapeutic interventions for COVID-19. By using icSHAPE, we determined the structural landscape of SARS-CoV-2 RNA in infected human cells and from refolded RNAs, as well as the regulatory untranslated regions of SARS-CoV-2 and six other coronaviruses. We validated several structural elements predicted in silico and discovered structural features that affect the translation and abundance of subgenomic viral RNAs in cells. The structural data informed a deep-learning tool to predict 42 host proteins that bind to SARS-CoV-2 RNA. Strikingly, antisense oligonucleotides targeting the structural elements and FDA-approved drugs inhibiting the SARS-CoV-2 RNA binding proteins dramatically reduced SARS-CoV-2 infection in cells derived from human liver and lung tumors. Our findings thus shed light on coronavirus and reveal multiple candidate therapeutics for COVID-19 treatment.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Reposicionamiento de Medicamentos , ARN Viral , Proteínas de Unión al ARN/antagonistas & inhibidores , SARS-CoV-2 , Animales , Línea Celular , Chlorocebus aethiops , Aprendizaje Profundo , Humanos , Conformación de Ácido Nucleico , ARN Viral/química , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genéticaRESUMEN
Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse range of double-stranded RNA (dsRNA) precursors to generate â¼22 nt microRNA (miRNA) or small interfering RNA (siRNA) products for sequence-directed gene silencing. In this work, we solved the cryoelectron microscopy (cryo-EM) structure of hDicer in complex with its cofactor protein TRBP and revealed the precise spatial arrangement of hDicer's multiple domains. We further solved structures of the hDicer-TRBP complex bound with pre-let-7 RNA in two distinct conformations. In combination with biochemical analysis, these structures reveal a property of the hDicer-TRBP complex to promote the stability of pre-miRNA's stem duplex in a pre-dicing state. These results provide insights into the mechanism of RNA processing by hDicer and illustrate the regulatory role of hDicer's N-terminal helicase domain.
Asunto(s)
ARN Helicasas DEAD-box/química , MicroARNs/química , Ribonucleasa III/química , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , MicroARNs/metabolismo , Coactivadores de Receptor Nuclear/química , Coactivadores de Receptor Nuclear/genética , Coactivadores de Receptor Nuclear/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , División del ARN , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.
Asunto(s)
Ficusina/química , ARN Bicatenario/química , Animales , Emparejamiento Base , Células HEK293 , Células HeLa , Humanos , Ratones , Células Madre Embrionarias de Ratones , ARN Largo no Codificante/químicaRESUMEN
Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.
Asunto(s)
Espectrometría de Masas/métodos , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Animales , Cromatina/metabolismo , Femenino , Silenciador del Gen , Humanos , Ratones , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/análisisRESUMEN
Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites1-4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.
Asunto(s)
Codón Iniciador , Conformación de Ácido Nucleico , ARN Bicatenario , ARN Mensajero , Humanos , Arabidopsis/genética , Arabidopsis/inmunología , Codón Iniciador/genética , Reconocimiento de Inmunidad Innata , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , Ribosomas/metabolismo , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Mensajero/genética , Transcriptoma , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Aprendizaje ProfundoRESUMEN
Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , ARN Largo no Codificante/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transcripción Genética , Animales , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Mutagénesis , Motivos de Nucleótidos , ARN Polimerasa II/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , ARN Largo no Codificante/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismoRESUMEN
Beyond transferring genetic information, RNAs are molecules with diverse functions that include catalyzing biochemical reactions and regulating gene expression. Most of these activities depend on RNAs' specific structures. Therefore, accurately determining RNA structure is integral to advancing our understanding of RNA functions. Here, we summarize the state-of-the-art experimental and computational technologies developed to evaluate RNA secondary and tertiary structures. We also highlight how the rapid increase of experimental data facilitates the integrative modeling approaches for better resolving RNA structures. Finally, we provide our thoughts on the latest advances and challenges in RNA structure determination methods, as well as on future directions for both experimental approaches and artificial intelligence-based computational tools to model RNA structure. Ultimately, we hope the technological advances will deepen our understanding of RNA biology and facilitate RNA structure-based biomedical research such as designing specific RNA structures for therapeutics and deploying RNA-targeting small-molecule drugs.
Asunto(s)
Biología Computacional , ARN , Inteligencia Artificial , Biología Computacional/métodos , Simulación por Computador , Modelos Moleculares , Conformación de Ácido Nucleico , ARN/química , ARN/genéticaRESUMEN
Fundamental to post-transcriptional regulation, the in vivo binding of RNA binding proteins (RBPs) on their RNA targets heavily depends on RNA structures. To date, most methods for RBP-RNA interaction prediction are based on RNA structures predicted from sequences, which do not consider the various intracellular environments and thus cannot predict cell type-specific RBP-RNA interactions. Here, we present a web server PrismNet that uses a deep learning tool to integrate in vivo RNA secondary structures measured by icSHAPE experiments with RBP binding site information from UV cross-linking and immunoprecipitation in the same cell lines to predict cell type-specific RBP-RNA interactions. Taking an RBP and an RNA region with sequential and structural information as input ('Sequence & Structure' mode), PrismNet outputs the binding probability of the RBP and this RNA region, together with a saliency map and a sequence-structure integrative motif. The web server is freely available at http://prismnetweb.zhanglab.net.
Asunto(s)
Proteínas de Unión al ARN , ARN , ARN/química , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Regulación de la Expresión GénicaRESUMEN
Transforming growth factor ß (TGF-ß) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-ß and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-ß and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.
Asunto(s)
ARN Largo no Codificante , Proteína smad7/metabolismo , Activinas/metabolismo , Activinas/farmacología , Animales , Diferenciación Celular , Ligandos , Ratones , ARN Largo no Codificante/metabolismo , Proteína smad7/genética , Proteína smad7/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) have emerged as significant players in almost every level of gene function and regulation. Thus, characterizing the structures and interactions of lncRNAs is essential for understanding their mechanistic roles in cells. Through a combination of (bio)chemical approaches and automated capillary and high-throughput sequencing (HTS), the complexity and diversity of RNA structures and interactions has been revealed in the transcriptomes of multiple species. These methods have uncovered important biological insights into the mechanistic and functional roles of lncRNA in gene expression and RNA metabolism, as well as in development and disease. In this review, we summarize the latest sequencing strategies to reveal RNA structure, RNA-RNA, RNA-DNA, and RNA-protein interactions, and highlight the recent applications of these approaches to map functional lncRNAs. We discuss the advantages and limitations of these strategies, and provide recommendations to further advance methodologies capable of mapping RNA structure and interactions in order to discover new biology of lncRNAs and decipher their molecular mechanisms and implication in diseases.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , Secuencia de Bases , ADN/química , HumanosRESUMEN
RNA folds to form diverse secondary and tertiary structures and often interacts with other biomolecules to function in cells. The technologies developed to map in vivo RNA structures and interactions can be broadly classified into four categories.
Asunto(s)
ARN/química , Análisis de Secuencia de ARN , ADN/química , Conformación de Ácido NucleicoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.
Asunto(s)
COVID-19/virología , Técnicas de Cultivo de Célula/métodos , SARS-CoV-2/fisiología , Antivirales/farmacología , Contención de Riesgos Biológicos , Genoma Viral/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/crecimiento & desarrollo , Replicación Viral/efectos de los fármacosRESUMEN
RNA molecules fold into complex structures that enable their diverse functions in cells. Recent revolutionary innovations in transcriptome-wide RNA structural probing of living cells have ushered in a new era in understanding RNA functions. Here, we summarize the latest technological advances for probing RNA secondary structures and discuss striking discoveries that have linked RNA regulation and biological processes through interrogation of RNA structures. In particular, we highlight how different long noncoding RNAs form into distinct secondary structures that determine their modes of interactions with protein partners to realize their unique functions. These dynamic structures mediate RNA regulatory functions through altering interactions with proteins and other RNAs. We also outline current methodological hurdles and speculate about future directions for development of the next generation of RNA structure-probing technologies of higher sensitivity and resolution, which could then be applied in increasingly physiologically relevant studies.
Asunto(s)
ARN/química , Animales , Humanos , Conformación de Ácido Nucleico , ARN/metabolismoRESUMEN
Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.
Asunto(s)
ARN Pequeño no Traducido/genética , ARN de Transferencia de Leucina/genética , Proteínas Ribosómicas/biosíntesis , Ribosomas/genética , Ribosomas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Emparejamiento Base , Secuencia de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Ratones , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Pequeño no Traducido/antagonistas & inhibidores , ARN de Transferencia de Leucina/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/efectos de los fármacos , Especificidad por Sustrato/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA molecules fold into complex structures that are important across many biological processes. Recent technological developments have enabled transcriptome-wide probing of RNA secondary structure using nucleases and chemical modifiers. These approaches have been widely applied to capture RNA secondary structure in many studies, but gathering and presenting such data from very different technologies in a comprehensive and accessible way has been challenging. Existing RNA structure probing databases usually focus on low-throughput or very specific datasets. Here, we present a comprehensive RNA structure probing database called RASP (RNA Atlas of Structure Probing) by collecting 161 deduplicated transcriptome-wide RNA secondary structure probing datasets from 38 papers. RASP covers 18 species across animals, plants, bacteria, fungi, and also viruses, and categorizes 18 experimental methods including DMS-seq, SHAPE-Seq, SHAPE-MaP, and icSHAPE, etc. Specially, RASP curates the up-to-date datasets of several RNA secondary structure probing studies for the RNA genome of SARS-CoV-2, the RNA virus that caused the on-going COVID-19 pandemic. RASP also provides a user-friendly interface to query, browse, and visualize RNA structure profiles, offering a shortcut to accessing RNA secondary structures grounded in experimental data. The database is freely available at http://rasp.zhanglab.net.
Asunto(s)
Biología Computacional/estadística & datos numéricos , Bases de Datos Genéticas/estadística & datos numéricos , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Conformación de Ácido Nucleico , ARN/química , Transcriptoma , Animales , COVID-19/epidemiología , COVID-19/prevención & control , COVID-19/virología , Biología Computacional/métodos , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Pandemias , ARN/genética , Sondas ARN/genética , ARN Bacteriano/química , ARN Bacteriano/genética , ARN de Hongos/química , ARN de Hongos/genética , ARN de Planta/química , ARN de Planta/genética , ARN Viral/química , ARN Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologíaRESUMEN
BACKGROUND: RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue. RESULTS: We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance. CONCLUSIONS: rG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.
Asunto(s)
G-Cuádruplex , Humanos , ARN/química , Transcriptoma , Células HEK293 , Análisis de Secuencia de ARN/métodosRESUMEN
SUMMARY: Structural visualization and analysis are fundamental to explore macromolecular functions. Here, we present a novel integrative web-based virtual reality (VR) system-VRmol, to visualize and study molecular structures in an immersive virtual environment. Importantly, it is integrated with multiple online databases and is able to couple structure studies with associated genomic variations and drug information in a visual interface by cloud-based drug docking. VRmol thus can serve as an integrative platform to aid structure-based translational research and drug design. AVAILABILITY AND IMPLEMENTATION: VRmol is freely available (https://VRmol.net), with detailed manual and tutorial (https://VRmol.net/docs). The code of VRmol is available as open source under the MIT license at http://github.com/kuixu/VRmol. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.