Frameshifting at collided ribosomes is modulated by elongation factor eEF3 and by integrated stress response regulators Gcn1 and Gcn20.
Frameshifting at collided ribosomes is modulated by elongation factor eEF3 and by integrated stress response regulators Gcn1 and Gcn20.
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DOI：
10.1261/rna.078964.121
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Grayhack EJ
中科院分区：
文献类型：
--
作者：
Houston L;Platten EM;Connelly SM;Wang J;Grayhack EJ
核糖体停滞可导致核糖体碰撞，从而引发质量控制反应，其功能之一是防止核糖体移码，这种活动需要保守的酵母蛋白 Mbf1 与 uS3 在碰撞核糖体上相互作用。然而，在核糖体碰撞过程中介导移码的全部因素尚不清楚。为了描述酿酒酵母中的此类因素，我们对影响从已知核糖体失速位点（CGA 密码子重复）移码的突变体进行了遗传选择。我们表明，一般翻译延伸因子 eEF3 和集成应激反应 (ISR) 途径成分 Gcn1 和 Gcn20 以相反的方式调节移码。我们发现了 eEF3 的突变形式，它可以特异性抑制移码，但不能抑制 CGA 密码子的翻译抑制。因此，我们推断碰撞核糖体处的移码需要 eEF3，它促进酵母和其他单细胞生物体中 tRNA-mRNA 易位和 E 位 tRNA 释放。相比之下，我们发现去除与 Mbf1 碰撞的核糖体结合的 Gcn1 或 Gcn20 会增加移码。因此，我们得出结论，移码被 Gcn1 和 Gcn20 抑制，尽管这些效应主要不是通过 ISR 的激活介导的。此外，我们检查了 eEF3 介导的移码与其他质量控制机制之间的关系，发现 Mbf1 需要 Hel2 或 Gcn1 来抑制野生型 eEF3 的移码。因此，这些结果提供了碰撞核糖体翻译延伸和移码之间直接联系的证据，以及移码受到作用于碰撞核糖体的质量控制机制限制的证据。
Ribosome stalls can result in ribosome collisions that elicit quality control responses, one function of which is to prevent ribosome frameshifting, an activity that entails the interaction of the conserved yeast protein Mbf1 with uS3 on colliding ribosomes. However, the full spectrum of factors that mediate frameshifting during ribosome collisions is unknown. To delineate such factors in the yeast Saccharomyces cerevisiae, we used genetic selections for mutants that affect frameshifting from a known ribosome stall site, CGA codon repeats. We show that the general translation elongation factor eEF3 and the integrated stress response (ISR) pathway components Gcn1 and Gcn20 modulate frameshifting in opposing manners. We found a mutant form of eEF3 that specifically suppressed frameshifting, but not translation inhibition by CGA codons. Thus, we infer that frameshifting at collided ribosomes requires eEF3, which facilitates tRNA–mRNA translocation and E-site tRNA release in yeast and other single cell organisms. In contrast, we found that removal of either Gcn1 or Gcn20, which bind collided ribosomes with Mbf1, increased frameshifting. Thus, we conclude that frameshifting is suppressed by Gcn1 and Gcn20, although these effects are not mediated primarily through activation of the ISR. Furthermore, we examined the relationship between eEF3-mediated frameshifting and other quality control mechanisms, finding that Mbf1 requires either Hel2 or Gcn1 to suppress frameshifting with wild-type eEF3. Thus, these results provide evidence of a direct link between translation elongation and frameshifting at collided ribosomes, as well as evidence that frameshifting is constrained by quality control mechanisms that act on collided ribosomes.
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