Regensburg Center for Biochemistry (RCB), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany.
Laboratory for Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
Nucleic Acids Res. 2020 Oct 9;48(18):10368-10382. doi: 10.1093/nar/gkaa704.
Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed.
环状 RNA（circRNAs）是一类广泛存在且保守的 RNA，由下游 5'到上游 3'剪接位点的反向剪接产生。circRNAs 具有组织特异性，并与包括癌症在内的多种疾病有关。它们可以作为 microRNAs（miRNAs）或 RNA 结合蛋白（RBPs）的海绵发挥作用，例如。此外，一些 circRNAs 含有开放阅读框（ORFs）并可能被翻译。然而，这些肽的功能相关性在很大程度上仍然难以捉摸。在这里，我们报告说，circZNF609 的 ORF 在表达 circZNF609 过表达构建体时可以有效地翻译。然而，内源性蛋白质无法检测到。此外，circZNF609 翻译的起始不依赖于产生 m6A 的酶 METTL3 或 RNA 序列元件，如内部核糖体进入位点（IRESs）。令人惊讶的是，全面的突变分析表明，缺乏产生 circZNF609 的缺失构建体仍会产生观察到的蛋白产物。这表明观察到的 circZNF609 翻译源自过表达质粒的反式剪接副产物，并强调需要仔细评估 circRNA 过表达构建体，特别是在进行功能研究时。
