RESUMO
Circular RNAs are garnering increasing interest as potential regulatory RNAs and a format for gene expression. The characterization of circular RNA using analytical techniques commonly employed in the literature, such as gel electrophoresis, can, under differing conditions, yield different results when attempting to distinguish circular RNA from linear RNA of similar molecular weights. Here, we describe circular RNA migration in different conditions, analyzed by gel electrophoresis and high-performance liquid chromatography (HPLC). We characterize key parameters that affect the migration pattern of circular RNA in gel electrophoresis systems, which include gel type, electrophoresis time, sample buffer composition, and voltage. Finally, we demonstrate the utility of orthogonal analytical tests for circular RNA that take advantage of its covalently closed structure to further distinguish circular RNA from linear RNA following in vitro synthesis.
Assuntos
RNA Circular , RNA , Eletroforese em Gel de Ágar/métodos , Peso Molecular , RNA/genética , RNA Circular/genéticaRESUMO
Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.
Assuntos
Proteína DEAD-box 58/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA/genética , Animais , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Imunidade Inata/genética , Camundongos , MicroRNAs/genética , RNA Circular , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Uridina/genética , Vacinas Sintéticas/genéticaRESUMO
The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and the presence of cancer cells with properties of normal tissue stem cells. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration. Here we show that mouse and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signalling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that these cells have features of normal tissue stem cells. Genetic perturbation of Wnt production or signalling suppressed tumour progression. Small-molecule inhibitors targeting essential posttranslational modification of Wnt reduced tumour growth and markedly decreased the proliferative potential of lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Progressão da Doença , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nicho de Células-Tronco , Proteínas Wnt/biossíntese , Via de Sinalização Wnt , Adenocarcinoma de Pulmão , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Taxa de Sobrevida , Proteínas Wnt/química , Proteínas Wnt/metabolismoRESUMO
Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.