Ikeuchi Ken, Inada Toshifumi
Graduate School of Pharmaceutical Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.
Sci Rep. 2016 Jun 17;6:28234. doi: 10.1038/srep28234.
Dom34-Hbs1 stimulates degradation of aberrant mRNAs lacking termination codons by dissociating ribosomes stalled at the 3' ends, and plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). In the dom34Δ mutant, nonstop mRNA is degraded by sequential endonucleolytic cleavages induced by a stalled ribosome at the 3' end. Here, we report that ribosome-associated Asc1/RACK1 is required for the endonucleolytic cleavage of nonstop mRNA by stalled ribosome at the 3' end of mRNA in dom34Δ mutant cells. Asc1/RACK1 facilitates degradation of truncated GFP-Rz mRNA in the absence of Dom34 and exosome-dependent decay. Asc1/RACK1 is required for the sequential endonucleolytic cleavages by the stalled ribosome in the dom34Δ mutant, depending on its ribosome-binding activity. The levels of peptidyl-tRNA derived from nonstop mRNA were elevated in dom34Δasc1Δ mutant cells, and overproduction of nonstop mRNA inhibited growth of mutant cells. E3 ubiquitin ligase Ltn1 degrades the arrest products from truncated GFP-Rz mRNA in dom34Δ and dom34Δasc1Δ mutant cells, and Asc1/RACK1 represses the levels of substrates for Ltn1-dependent degradation. These indicate that ribosome-associated Asc1/RACK1 facilitates endonucleolytic cleavage of nonstop mRNA by stalled ribosomes and represses the levels of aberrant products even in the absence of Dom34. We propose that Asc1/RACK1 acts as a fail-safe in quality control for nonstop mRNA.
Dom34-Hbs1通过解离停滞在3'端的核糖体来刺激缺乏终止密码子的异常mRNA的降解，在无义介导的mRNA降解（NSD）和无-go降解（NGD）中发挥关键作用。在dom34Δ突变体中，无义mRNA通过3'端停滞的核糖体诱导的顺序内切核酸酶切割而降解。在这里，我们报告核糖体相关的Asc1/RACK1是dom34Δ突变体细胞中3'端停滞的核糖体对无义mRNA进行内切核酸酶切割所必需的。在没有Dom34和外泌体依赖性降解的情况下，Asc1/RACK1促进截短的GFP-Rz mRNA降解。Asc1/RACK1是dom34Δ突变体中停滞核糖体进行顺序内切核酸酶切割所必需的，这取决于其核糖体结合活性。在dom34Δasc1Δ突变体细胞中，源自无义mRNA的肽基-tRNA水平升高，无义mRNA的过量产生抑制突变体细胞的生长。E3泛素连接酶Ltn1在dom34Δ和dom34Δasc1Δ突变体细胞中降解截短的GFP-Rz mRNA的停滞产物，并且Asc1/RACK1抑制Ltn1依赖性降解的底物水平。这些表明核糖体相关的Asc1/RACK1促进停滞核糖体对无义mRNA的内切核酸酶切割，并且即使在没有Dom34的情况下也抑制异常产物的水平。我们提出Asc1/RACK1在无义mRNA的质量控制中起安全保障作用。
