School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland; Science Foundation Center for Research Training in Genomics Data Science, University College Cork, Cork, Ireland.
Mol Cell. 2022 Oct 6;82(19):3745-3749.e2. doi: 10.1016/j.molcel.2022.08.024. Epub 2022 Sep 16.
The research article describing the discovery of ribosomal frameshifting in the bacterial CopA gene also reported the occurrence of frameshifting in the expression of the human ortholog ATP7B based on assays using dual luciferase reporters. An examination of the publicly available ribosome profiling data and the phylogenetic analysis of the proposed frameshifting site cast doubt on the validity of this claim and prompted us to reexamine the evidence. We observed similar apparent frameshifting efficiencies as the original authors using the same type of vector that synthesizes both luciferases as a single polyprotein. However, we noticed anomalously low absolute luciferase activities from the N-terminal reporter that suggests interference of reporter activity or levels by the ATP7B test cassette. When we tested the same proposed ATP7B frameshifting cassette in a more recently developed reporter system in which the reporters are released without being included in a polyprotein, no frameshifting was detected above background levels.
描述细菌 CopA 基因中核糖体移码发现的研究文章还报告了基于使用双荧光素酶报告基因的测定，在人类同源物 ATP7B 的表达中发生移码。对公开可用的核糖体分析数据的检查和对提议的移码位点的系统发育分析对这一说法的有效性提出了质疑，并促使我们重新检查证据。我们使用与原始作者相同的类型的载体观察到了类似的明显移码效率，该载体将两种荧光素酶合成到一个多蛋白中。然而，我们注意到来自 N 端报告者的异常低的绝对荧光素酶活性，这表明报告者活性或水平受到 ATP7B 测试盒的干扰。当我们在最近开发的报告系统中测试相同的拟议 ATP7B 移码盒时，在没有包含在多蛋白中的情况下释放报告者，在背景水平以上没有检测到移码。
