Time-Resolved Visualization of the Yeast Ribosome-Biogenesis by Cryo-Electron Tomography

通过冷冻电子断层扫描对酵母核糖体生物发生进行时间分辨可视化

• 批准号: 400861327
• 负责人: Professor Dr. Achilleas Frangakis
• 金额: --
• 依托单位: Institut für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/400861327?language=en
• 关键词: Time; Resolved; Visualization; Yeast; Ribosome

In this project, we would like to analyze the origins of yeast ribosome biogenesis using cryo-electron tomography (cryo-ET) and correlative light and electron microscopy (CLEM). The ribosome plays a central role in gene expression. It translates the genetic information that is transcribed within the messenger RNA (mRNA) to the proteome of the cell. The importance of the ribosome and the complexity of its structure necessitate a well-coordinated and regulated assembly. Malfunctions of this process are mostly lethal for the cell and can cause severe pathology.While the mature ribosome is well studied, the structural understanding of its biogenesis remains elusive. Eukaryotic ribosome biogenesis starts in the nucleolus with the synthesis of a precursor of the ribosomal RNA. A plethora of ribosome-assembly factors associate with this RNA precursor forming pre-ribosomal particles. We recently visualized these pre-ribosomal particles in the context of the so-called Miller trees (Neyer et al., Nature 2016). Miller trees are supramolecular structures with a ribosomal DNA (rDNA) scaffold that contain actively transcribing RNA polymerase I (Pol I) enzymes, from which the nascent ribosomal RNA (rRNA) chain is emerging and folds to form the pre-ribosomal particles. These pre-ribosomal particles are located at the end of the branches of the Miller trees and are referred to as terminal knobs. Thus, the terminal knobs of the Miller trees visualize the stepwise generation of pre-ribosomes in a quasi-sequential manner. With this application, we would like to analyze the terminal knobs that form co-transcriptionally at the 5’-end of the emerging rRNA through the stepwise addition of protein complexes. Such an analysis approach is only feasible due to the defined localization of the co-transcriptionally assembling complexes on the growing pre-rRNAs of the Miller trees. In yeast, the formation of the terminal knobs is a two-stage process: Early formed knobs will be cleaved from the nascent rRNA chain to form the small ribosomal subunit and are therefore called small subunit (SSU) processome. Later formed knobs will develop to the large ribosomal subunit and are therefore called large subunit (LSU) processome. Thus, the knob generation is a two-stage process that represents the earliest precursors of the final ribosomal subunits. Due to their size (which is approximately 6 MDa for the SSU processome), cryo-electron tomography is ideally suited for the analysis of the terminal knobs. Further on specific labeling of defined states can precisely localize distinct states and facilitate our analysis. Ultimately, this study should result in a more detailed understanding of the structure and function of the SSU and LSU processomes, and provide unprecedented insights into the fascinating mechanism of the early ribosome biogenesis in vivo.

在这个项目中，我们希望使用冷冻电子断层扫描（cryo-ET）和相关光电子显微镜（CLEM）来分析酵母核糖体生物发生的起源核糖体在基因表达中发挥着核心作用。核糖体的重要性及其结构的复杂性需要良好的协调和调节。该过程的故障对细胞来说大多是致命的，并可能导致严重的病理。虽然成熟核糖体已得到充分研究，但对其生物发生的结构理解仍然难以捉摸。真核核糖体的生物发生始于核仁的前体合成。大量核糖体组装因子与形成前核糖体颗粒相关。所谓的米勒树（Neyer et al., Nature 2016）是具有核糖体 DNA (rDNA) 支架的超分子结构，其中包含主动转录 RNA 聚合酶 I (Pol I)，从中产生新生核糖体 RNA (rRNA)。链正在出现并折叠形成前核糖体颗粒，这些前核糖体颗粒位于米勒树分支的末端，被称为末端。因此，米勒树的末端旋钮以准顺序方式可视化前核糖体的逐步生成，我们希望分析在 5' 端共转录形成的末端旋钮。通过逐步添加蛋白质复合物来分析新兴的 rRNA，这种分析方法只有在米勒树生长的前 rRNA 上共转录组装复合物的明确定位时才可行。在酵母中，末端旋钮的形成是一个两阶段的过程：早期形成的旋钮将从新生的 rRNA 链上裂解，形成小核糖体亚基，因此称为小亚基（SSU）加工组。大核糖体亚基，因此被称为大亚基（LSU）加工组，因此，旋钮的生成是一个两阶段的过程，由于它们的存在，它代表了最终核糖体亚基的最早前体。由于尺寸（SSU 处理组的大小约为 6 MDa），冷冻电子断层扫描非常适合分析末端旋钮。此外，对定义状态的特定标记可以精确定位不同的状态并促进我们的分析。使人们对 SSU 和 LSU 过程组的结构和功能有了更详细的了解，并为体内早期核糖体生物发生的迷人机制提供了前所未有的见解。