Meganck Rita M, Liu Jiacheng, Hale Andrew E, Simon Katherine E, Fanous Marco M, Vincent Heather A, Wilusz Jeremy E, Moorman Nathaniel J, Marzluff William F, Asokan Aravind
Curriculum in Genetics & Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Surgery, Duke University School of Medicine, Durham, NC 27708, USA.
Mol Ther Nucleic Acids. 2021 Jan 16;23:821-834. doi: 10.1016/j.omtn.2021.01.003. eCollection 2021 Mar 5.
Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for expression of therapeutic proteins and non-coding RNAs. In eukaryotes, circRNAs are primarily generated by the spliceosome through backsplicing. Here, we interrogate different molecular elements including intron type and length, repeats, internal ribosome entry sites (IRESs), and exon length essential for circRNA formation and exploit this information to engineer robust backsplicing and circRNA expression. Specifically, we leverage the finding that the downstream intron can tolerate large inserts without affecting splicing to achieve tandem expression of backspliced circRNAs and tRNA intronic circRNAs from the same template. Further, truncation of selected intronic regions markedly increased circRNA formation in different cell types as well as AAV-mediated circRNA expression in cardiac and skeletal muscle tissue . We also observed that different IRES elements and exon length influenced circRNA expression and translation, revealing an exonic contribution to splicing, as evidenced by different RNA species produced. Taken together, these data provide new insight into improving the design and expression of synthetic circRNAs. When combined with AAV capsid and promoter technologies, the backsplicing introns and IRES elements constituting this modular platform significantly expand the gene expression toolkit.
环状RNA（circRNAs）是高度稳定的RNA分子，是表达治疗性蛋白质和非编码RNA的理想模板。在真核生物中，circRNAs主要由剪接体通过反向剪接产生。在此，我们研究了不同的分子元件，包括内含子类型和长度、重复序列、内部核糖体进入位点（IRESs）以及circRNA形成所必需的外显子长度，并利用这些信息来设计强大的反向剪接和circRNA表达。具体而言，我们利用下游内含子能够耐受大的插入片段而不影响剪接这一发现，实现了从同一模板串联表达反向剪接的circRNAs和tRNA内含子circRNAs。此外，对选定内含子区域的截短显著增加了不同细胞类型中circRNA的形成，以及心脏和骨骼肌组织中AAV介导的circRNA表达。我们还观察到不同的IRES元件和外显子长度会影响circRNA的表达和翻译，这揭示了外显子对剪接的贡献，不同RNA种类的产生就是证明。综上所述，这些数据为改进合成circRNAs的设计和表达提供了新的见解。当与AAV衣壳和启动子技术相结合时，构成这个模块化平台的反向剪接内含子和IRES元件显著扩展了基因表达工具包。
