Wellcome Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh, EH9 3BF, Scotland.
Institute of Technology, University of Tartu, Nooruse 150411, Tartu, Estonia.
Nat Commun. 2019 Feb 4;10(1):563. doi: 10.1038/s41467-019-08382-z.
Ribosome-associated quality control (RQC) pathways monitor and respond to ribosome stalling. Using in vivo UV-crosslinking and mass spectrometry, we identified a C-terminal region in Hel2/Rqt1 as an RNA binding domain. Complementary crosslinking and sequencing data for Hel2 revealed binding to 18S rRNA and translated mRNAs. Hel2 preferentially bound mRNAs upstream and downstream of the stop codon. C-terminal truncation of Hel2 abolished the major 18S crosslink and polysome association, and altered mRNA binding. HEL2 deletion caused loss of RQC and, we report here, no-go decay (NGD), with comparable effects for Hel2 truncation including the RNA-binding site. Asc1 acts upstream of Hel2 in RQC and asc1∆ impaired Hel2 binding to 18S and mRNA. In conclusion: Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. This 18S interaction is required for Hel2 function in RQC and NGD. Hel2 probably interacts with mRNA during translation termination.
核糖体相关质量控制 (RQC) 途径监测和响应核糖体停滞。通过体内 UV 交联和质谱分析，我们鉴定出 Hel2/Rqt1 的 C 端区域是一个 RNA 结合结构域。互补交联和测序数据表明 Hel2 与 18S rRNA 和翻译的 mRNA 结合。Hel2 优先结合终止密码子上下游的 mRNA。Hel2 的 C 端截断消除了主要的 18S 交联和多核糖体结合，并改变了 mRNA 结合。HEL2 的缺失导致 RQC 丧失，我们在这里报告无意义衰变 (NGD)，Hel2 截断包括 RNA 结合位点也有类似的影响。Asc1 在 RQC 中作用于 Hel2 的上游，asc1∆ 损害了 Hel2 与 18S 和 mRNA 的结合。总之：Hel2 通过与 18S rRNA 和 Asc1 的相互作用被招募或稳定在翻译的 40S 核糖体亚基上。这种 18S 相互作用是 Hel2 在 RQC 和 NGD 中发挥作用所必需的。Hel2 可能在翻译终止过程中与 mRNA 相互作用。
