Identification of the mRNA targets of tRNA-specific regulation using genome-wide simulation of translation.
Identification of the mRNA targets of tRNA-specific regulation using genome-wide simulation of translation.
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DOI：
10.1093/nar/gkw630
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发表时间：
2016-11-02
影响因子：
14.9
通讯作者：
Stansfield I
中科院分区：
文献类型：
--
作者：
Gorgoni B;Ciandrini L;McFarland MR;Romano MC;Stansfield I
tRNA基因拷贝数是tRNA丰度的主要决定因素，因此是每个tRNA在翻译过程中向核糖体递送氨基酸的速率。低丰度的tRNA对稀有密码子的解码缓慢，但目前还不清楚哪些基因可能受到tRNA介导的表达调控。在这里，这些mRNA靶点通过翻译的全局模拟来鉴定。对野生型和sup 70 -65突变体中的每种mRNA进行了计算机模拟mRNA翻译速率比较，sup 70 -65突变体表现出假菌丝生长表型和75%较慢的CAG密码子翻译速率。在4900个含CAG的mRNA中，300个在模拟的tRNA突变体中显示出显著降低的计算机翻译速率。定量免疫分析证实敏感mRNA的翻译速率降低具有浓度依赖性。翻译模拟表明，降低浓度触发核糖体队列，这消散在降低翻译起始速率。为了验证这一预测实验，组成型gcn 2激酶突变体被用来降低体内翻译起始速率。这修复了目标mRNA在sup 70 -65背景下的相对翻译速率缺陷，并改善了sup 70 -65假菌丝生长表型。因此，我们验证了全球模拟翻译作为一种新的工具，以确定mRNA的目标tRNA特异性基因调控。
tRNA gene copy number is a primary determinant of tRNA abundance and therefore the rate at which each tRNA delivers amino acids to the ribosome during translation. Low-abundance tRNAs decode rare codons slowly, but it is unclear which genes might be subject to tRNA-mediated regulation of expression. Here, those mRNA targets were identified via global simulation of translation. In-silico mRNA translation rates were compared for each mRNA in both wild-type and a sup70-65 mutant, which exhibits a pseudohyphal growth phenotype and a 75% slower CAG codon translation rate. Of 4900 CAG-containing mRNAs, 300 showed significantly reduced in silico translation rates in a simulated tRNA mutant. Quantitative immunoassay confirmed that the reduced translation rates of sensitive mRNAs were concentration-dependent. Translation simulations showed that reduced concentrations triggered ribosome queues, which dissipated at reduced translation initiation rates. To validate this prediction experimentally, constitutive gcn2 kinase mutants were used to reduce in vivo translation initiation rates. This repaired the relative translational rate defect of target mRNAs in the sup70-65 background, and ameliorated sup70-65 pseudohyphal growth phenotypes. We thus validate global simulation of translation as a new tool to identify mRNA targets of tRNA-specific gene regulation.
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通讯作者：
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