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 read-through 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 (pre-mature 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 read-through. 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 read-through. 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 read-through events.

中文翻译：

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酿酒酵母中假尿苷介导的终止密码子通读与序列上下文无关

我们之前已经表明，当终止密码子（UAA、UAG 或 UGA）的尿苷被假尿苷化时，核糖体会读取修改后的终止密码子。然而，尚不清楚假尿苷 (Ψ) 介导的通读是否取决于 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，当终止密码子（过早终止密码子，PTC）在几个不同的位置（具有不同的序列背景）被引入到报告基因的编码区并假尿苷化时，我们检测到相似的通读水平。使用突变和选择/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（上游和下游）在 Ψ 介导的核糖体通读中发挥的作用很小（如果有的话）。有趣的是，我们没有检测到酵母系统中靶向 PTC 假尿苷化对 NMD（无义介导的 mRNA 衰减）的抑制。我们的结果表明 Ψ 介导的无义抑制发生在翻译水平，并且这种抑制与序列上下文无关，这与之前描述的一些罕见的终止密码子通读事件不同。