RESUMEN
Circular RNAs (circRNAs) are noncoding RNAs that exist in all eukaryotes investigated and are derived from back-splicing of certain pre-mRNA exons. Here, we report the application of artificial circRNAs designed to act as antisense-RNAs. We systematically tested a series of antisense-circRNAs targeted to the SARS-CoV-2 genome RNA, in particular its structurally conserved 5'-untranslated region. Functional assays with both reporter transfections as well as with SARS-CoV-2 infections revealed that specific segments of the SARS-CoV-2 5'-untranslated region can be efficiently accessed by specific antisense-circRNAs, resulting in up to 90% reduction of virus proliferation in cell culture, and with a durability of at least 48 h. Presenting the antisense sequence within a circRNA clearly proved more efficient than in the corresponding linear configuration and is superior to modified antisense oligonucleotides. The activity of the antisense-circRNA is surprisingly robust towards point mutations in the target sequence. This strategy opens up novel applications for designer circRNAs and promising therapeutic strategies in molecular medicine.
Asunto(s)
Genoma Viral/genética , ARN sin Sentido/genética , ARN Circular/genética , ARN Viral/genética , SARS-CoV-2/genética , Replicación Viral/genética , Regiones no Traducidas 5'/genética , Animales , Antivirales/metabolismo , Secuencia de Bases , COVID-19/prevención & control , COVID-19/virología , Proliferación Celular/genética , Chlorocebus aethiops , Diseño de Fármacos , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Conformación de Ácido Nucleico , ARN Viral/química , RNA-Seq/métodos , SARS-CoV-2/fisiología , Células VeroRESUMEN
Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.
Asunto(s)
Vesículas Extracelulares/metabolismo , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Línea Celular , Vesículas Extracelulares/ultraestructura , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Humanos , Poli A/metabolismo , Poliadenilación , Caperuzas de ARN/metabolismo , ARN Polimerasa III/metabolismo , ARN Mensajero/genéticaRESUMEN
Circular RNAs (circRNAs) have been studied extensively in the last few years, uncovering functional roles in a diverse range of cell types and organisms. As shown for a few cases, these functions may be mediated by trans-acting factors, in particular RNA-binding proteins (RBPs). However, the specific interaction partners for most circRNAs remain unknown. This is mainly due to technical difficulties in their identification and in differentiating between interactors of circRNAs and their linear counterparts. Here we review the currently used methodology to systematically study circRNA-protein complexes (circRNPs), focusing either on a specific RNA or protein, both on the gene-specific or global level, and discuss advantages and challenges of the available approaches.
Asunto(s)
ARN Circular , ARN , ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismoRESUMEN
Non-coding RNAs were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs) were only recently discovered as a new large group of non-coding RNAs that, due to their circular configuration, are metabolically more stable compared to their linear counterparts and therefore highly suitable for biomarker use. Based on high-throughput sequencing, the catalogs of endogenous circRNAs with disease relevance and correlation continue to grow steadily. As a consequence, circRNAs emerged as novel and attractive biomarkers, indicated by numerous recent publications. Here we would like to stress the need of essential quality criteria for validation and characterization of circular RNAs. In addition to high-throughput sequencing, classical biochemical methods are essential and should be applied for the characterization of this special class of RNAs, in particular to convincingly confirm their circularity.