Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, New York 14642, USA.
RNA. 2020 Sep;26(9):1247-1256. doi: 10.1261/rna.076042.120. Epub 2020 May 20.
We have previously shown that when the uridine of a stop codon (UAA, UAG, or UGA) is pseudouridylated, the ribosome reads through the modified stop codon. However, it is not clear as to whether or not the pseudouridine (Ψ)-mediated readthrough is dependent on the sequence context of mRNA. Here, we use several different approaches and the yeast system to address this question. We show that when a stop codon (premature termination codon, PTC) is introduced into the coding region of a reporter mRNA at several different positions (with different sequence contexts) and pseudouridylated, we detect similar levels of readthrough. Using mutational and selection/screen analyses, we also show that the upstream sequence (relative to PTC) as well as the nucleotides surrounding the PTC (upstream and downstream) play a minimal role (if at all) in Ψ-mediated ribosome readthrough. Interestingly, we detect no suppression of NMD (nonsense-mediated mRNA decay) by targeted PTC pseudouridylation in the yeast system. Our results indicate that Ψ-mediated nonsense suppression occurs at the translational level, and that the suppression is sequence context-independent, unlike some previously characterized rare stop codon readthrough events.
我们之前已经证明，当终止密码子（UAA、UAG 或 UGA）的尿嘧啶被假尿嘧啶化时，核糖体可以通读修饰后的终止密码子。然而，目前尚不清楚假尿嘧啶（Ψ）介导的通读是否依赖于 mRNA 的序列背景。在这里，我们使用几种不同的方法和酵母系统来解决这个问题。我们表明，当一个终止密码子（过早终止密码子，PTC）在报告 mRNA 的编码区域的几个不同位置（具有不同的序列背景）被引入并被假尿嘧啶化时，我们检测到类似水平的通读。通过突变和选择/筛选分析，我们还表明，上游序列（相对于 PTC）以及 PTC 周围的核苷酸（上下游）在 Ψ 介导的核糖体通读中作用最小（如果有的话）。有趣的是，我们在酵母系统中没有检测到靶向 PTC 假尿嘧啶化对 NMD（无意义介导的 mRNA 降解）的抑制。我们的结果表明，Ψ 介导的无意义抑制发生在翻译水平，并且抑制是序列上下文无关的，与一些先前表征的罕见终止密码子通读事件不同。
