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

在本计画中，我们将利用冷冻电子断层摄影术（cryo-ET）及相关光学与电子显微镜（CLEM）来分析酵母核糖体生物合成的起源。核糖体在基因表达中起核心作用。它将信使RNA（mRNA）内转录的遗传信息翻译为细胞的蛋白质组。核糖体的重要性及其结构的复杂性需要一个协调和调节良好的组装。这一过程的异常对细胞来说是致命的，并可能导致严重的病理。虽然成熟的核糖体已被很好地研究，但对其生物起源的结构理解仍然难以捉摸。真核生物核糖体的生物发生始于核仁中核糖体RNA前体的合成。大量的核糖体组装因子与这种RNA前体相关，形成前核糖体颗粒。我们最近在所谓的米勒树（Neyer et al.，Nature 2016）。米勒树是具有核糖体DNA（rDNA）支架的超分子结构，所述核糖体DNA（rDNA）支架含有活跃转录的RNA聚合酶I（Pol I）酶，新生的核糖体RNA（rRNA）链从所述支架出现并折叠以形成前核糖体颗粒。这些前核糖体颗粒位于米勒树分支的末端，被称为末端结。因此，米勒树的末端结以准顺序的方式可视化前核糖体的逐步生成。通过该应用，我们希望分析通过逐步添加蛋白质复合物在新兴rRNA的5 '端共转录形成的末端结。这样的分析方法是唯一可行的，因为共转录组装复合物在米勒树的生长前体rRNA上的确定的定位。在酵母中，末端结的形成是一个两阶段的过程：早期形成的结将从新生的rRNA链上切割下来，形成小的核糖体亚基，因此被称为小亚基（SSU）加工体。后来形成的球将发展成大亚基，因此被称为大亚基（LSU）加工体。因此，结的产生是一个两阶段的过程，代表了最终核糖体亚基的最早的前体。由于其尺寸（SSU处理器约为6 MDa），低温电子断层扫描非常适合分析终端旋钮。此外，对定义的状态进行特定的标记可以精确地定位不同的状态，便于我们的分析。最终，这项研究将导致对SSU和LSU加工体的结构和功能的更详细的了解，并为体内早期核糖体生物发生的迷人机制提供前所未有的见解。

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.