Mechanism of stringent translation initiation: a probe for its biological relevance

真核生物翻译起始是一个复杂的过程，涉及核糖体、mRNA、Met-tRNAiMet 和许多真核起始因子（eIFs）。数十年的研究， 模式真核生物酿酒酵母揭示，关键过程是形成 密码子-反密码子碱基配对在小核糖体P位点。严格的启动是通过 严格在AUG起始密码子处形成48 S核糖体前起始复合物（PIC）， 不包括在其他研究中心启动。然而，有趣的是，许多非规范的起始位点被利用 在某些生物学背景和疾病如癌症和神经退行性疾病中。的 人类基因组中的非AUG起始位点的列表远未完成，如何使用 因此，这些位点的蛋白质生产受到调控， 问题 因此，该基金的目标1是通过全基因组研究， 翻译谱的良好表征的癌症模型系统，验证这些网站中的一些 并确定在癌症中驱动观察到的非AUG翻译调节的机制。 目的二是研究5 MP和Met-tRNAiMet腺苷N6- 苏氨酰氨甲酰化（t6 A）在控制非AUG翻译。将测试是否为5 MP 在许多类型的癌症中发现的突变可以改变起始准确性，从而影响患者的预后。 预后我们的初步研究表明，位于Met-tRNAiMet反密码子3'端的t6 A可以通过抑制细胞凋亡来抑制细胞凋亡。 与eIFl相比，eIFl特异性地区分GUG和UUG起始密码子， universal non-AUG结合分子动力学模拟方法，我们将测试 如果最近发现的循环t6 A起到区分作用，并确定 t6 A和eIF 1之间的合作或竞争促进严格起始和漏扫描 对翻译调控至关重要。 目的3是研究起始密码子选择的独特机制， 在热休克反应期间在平移水平。这一机制被发现 通过酵母eIF 3 i突变体的翻译谱分析， eIF 3g亚基在高温下。工作假设假设，在高温下， 在AUG密码子处稳定的PIC形成需要额外的mRNA元件锚定 在其进入位点启动核糖体。这些假设的mRNA元件位于下游 并包括一个新的eIF 3g-结合基序5 '-GUCG-3'和一个下游茎- 潜在结合40 S的进入位点侧的环，从而稳定PIC形成。 该模型将使用酵母作为模型系统进行测试。

Eukaryotic translation initiation is a complex process involving the ribosome, mRNA, Met-tRNAiMet and numerous eukaryotic initiation factors (eIFs). Decades of studies driven by those using the model eukaryote yeast Saccharomyces cerevisiae revealed that the key process is the formation of codon-anticodon base pairing in the small ribosome P-site. Stringent initiation is enabled by formation of the 48S ribosomal pre-initiation complex (PIC) strictly at the AUG start codon while excluding initiation at other sites. Intriguingly, however, many non-canonical start sites are utilized in some biological contexts and diseases such as cancer and neurodegenerative disorders. The list of non-AUG start sites within the human genome is far from being complete and how the use of these sites and hence protein production from these sites are regulated remains an open question. Thus, Aim 1 of this grant is to make such a list of non-AUG start sites through genome-wide translation profiling of well characterized cancer model systems, verify some of these sites and determine the mechanism driving the observed non-AUG translational regulation in cancer. The Aim 2 is to study the mechanistic role of 5MP and Met-tRNAiMet adenosine N6- threonylcarbamoylation (t6A) in controlling non-AUG translation. It will be tested if 5MP mutations found in many types of cancer can alter initiation accuracy, thereby affecting patients' prognosis. Our preliminary studies suggested that t6A located 3' of Met-tRNAiMet anticodon can discriminate specifically against GUG and UUG start codons, in contrast to eIF1 being more universal non-AUG discriminator. Combining molecular dynamics simulation methods, we will test if the recently discovered cyclic t6A serves the discriminating role and determine how the cooperation or competition between t6A and eIF1 promotes stringent initiation and leaky scanning crucial for translational regulation. The Aim 3 is to study yet a distinct mechanism of start codon selection that is exploited during the heat shock response at the translational level. This mechanism was discovered through translational profiling of yeast eIF3i mutant defective in its interaction with RNA-binding eIF3g subunit at a high temperature. The working hypothesis assumes that, at a high temperature, stable PIC formation at the AUG codon requires additional mRNA elements anchoring the initiating ribosome at its entry site. These hypothetical mRNA elements are located downstream of the start codon and include a novel eIF3g-binding motif 5'-GUCG-3' and a downstream stem- loop that potentially binds the entry site-side of the 40S, thereby stabilizing the PIC formation. This model will tested using yeast as a model system.