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

真核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（eIF）。由那些使用该技术的人推动的数十年的研究 模型真核酵母酿酒酵母揭示关键过程是形成 小核糖体 P 位点的密码子-反密码子碱基配对。严格启动是通过 严格在 AUG 起始密码子处形成 48S 核糖体预起始复合物 (PIC)，同时 不包括在其他地点启动。然而有趣的是，许多非规范的起始站点被利用 在某些生物学背景和疾病中，例如癌症和神经退行性疾病。的 人类基因组内的非 AUG 起始位点列表还远未完成，以及如何使用 这些位点的数量以及因此这些位点的蛋白质生产是否受到调节仍然是一个开放的问题 问题。 因此，本次资助的目标 1 是通过全基因组范围制作这样一个非 AUG 起始位点列表 对特征明确的癌症模型系统进行翻译分析，验证其中一些位点 并确定驱动癌症中观察到的非 AUG 翻译调节的机制。 目标 2 是研究 5MP 和 Met-tRNAiMet 腺苷 N6- 的机制作用 苏氨酰氨基甲酰化 (t6A) 在控制非 AUG 翻译中的作用。将测试是否为5MP 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。我们的初步研究表明位于 Met-tRNAiMet 反密码子 3' 的 t6A 可以 专门区分 GUG 和 UUG 起始密码子，与 eIF1 相比 通用非 AUG 鉴别器。结合分子动力学模拟方法，我们将测试 如果最近发现的环状 t6A 起到区分作用并确定如何 t6A和eIF1之间的合作或竞争促进严格启动和泄漏扫描 对于翻译调控至关重要。 目标 3 是研究一种独特的起始密码子选择机制 在平移水平的热休克反应期间。这个机制被发现了 通过对与 RNA 结合相互作用存在缺陷的酵母 eIF3i 突变体进行翻译分析 eIF3g 亚基在高温下。工作假设假设，在高温下， 在 AUG 密码子处形成稳定的 PIC 需要额外的 mRNA 元件来锚定 在其进入位点启动核糖体。这些假设的 mRNA 元件位于下游 起始密码子，包括一个新的 eIF3g 结合基序 5'-GUCG-3' 和一个下游茎- 环可能结合 40S 的入口位点侧，从而稳定 PIC 的形成。 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.