Ultrastructural Analysis of RNA Synthesis and Processing

0094561安·拜尔该项目的长期目标是帮助理解核糖体生物发生的复杂过程。核糖体作为细胞的蛋白质合成机制具有明显的意义和重要性。在活跃生长的酵母细胞(酿酒酵母)中，核糖体以每分钟约2000的速度制造，这个过程被证明是令人惊讶的复杂的。事实上，已知有60多种反式作用成分在酵母核糖体生物发生中发挥作用，还没有证明可以从纯化的成分组装核糖体。这项研究将为核糖体合成的早期事件提供一个新的视角，这些事件对于成功组装成熟的核糖体非常重要。该项目结合了两种以前从未同时开发的强大方法。具体地说，它们是酵母遗传学和米勒染色质铺展技术，用于电子显微镜(EM)显示活性基因。基本的想法是在酵母细胞中可视化核糖体RNA基因，这些基因在核糖体RNA加工的早期步骤中的反式作用因子中具有突变。这三个最大的rRNA是由RNA聚合酶I合成的，作为单一的大的35S前体RNA，并被广泛的修饰(主要通过假脲和核糖甲基化)和通过核溶解切割分离。在合成这种rRNA所需的5-6分钟内，新生的转录本可以在典型的重复的‘圣诞树’基因构型中进行EM可视化。这些rRNA转录本被包装成大的核糖核蛋白复合体，出现在非随机位置。通过询问随着各种成分的缺失或突变，这种RNP结构是否以及如何变化，结合关于突变对rRNA加工的影响的知识，将获得有关加工的结构框架、加工中的分子相互作用以及这些事件的时间和顺序的信息。此外，三个rRNA加工事件可以低频率但可重复性地共转录可视化。所看到的三个事件被认为代表了转录本的极端5‘端(5’ETS)，大部分转录本从极端3‘端(3’ETS)分离，以及转录本中途的切割，这可能对应于ITS1中的切割，将18S rRNA前体与前体的其余部分分开。这些预测将使用已知必需成分的突变进行测试，如果正确，将在结构设置中测试对这些特定处理步骤的影响。待检测的成分包括rRNA修饰所涉及的两类主要snoRNP(框H/ACA和框C+D)的RNA和蛋白质成分，重点是早期RNA切割步骤所需的snoRNP，以及参与这些步骤的非snoRNP蛋白。这些实验的完成将为核糖体生物发生的早期步骤提供重要的新信息。

0094561 Ann Beyer The long term goal of the project is to contribute to an understanding of the complex process of ribosome biogenesis. Ribosomes are of obvious interest and importance as the protein synthesis machinery of the cell. In an actively growing yeast cell (Saccharomyces cerevisiae), ribosomes are made at a rate of ~2000 per minute in a process that has turned out to be surprisingly complex. Indeed, more than 60 trans-acting components are known to function in yeast ribosome biogenesis, and it has not proven possible to assemble ribosomes from purified components. This research will provide a new perspective on early events in ribosome synthesis, which are important to the successful assembly of the mature ribosome. The project combines two powerful approaches that have not previously been exploited simultaneously. Specifically, these are yeast genetics and the Miller chromatin spreading technique for electron microscope (EM) visualization of active genes. The basic idea is to visualize ribosomal RNA genes in yeast cells that have mutations in the trans-acting factors that function in early steps in ribosomal RNA processing. The three largest rRNAs are synthesized by RNA polymerase I as a single large 35S precursor RNA and are extensively modified (mainly by pseudouridylation and ribose methylation) and separated by nucleolytic cleavages. During the 5-6 minutes required to synthesize this rRNA, the nascent transcripts are accessible for EM visualization in the characteristic tandemly repeated 'Christmas-tree' gene configuration. These rRNA transcripts are packaged into large ribonucleoprotein complexes that occur at nonrandom positions. By asking if and how this RNP structure changes as various components are deleted or mutated, combined with knowledge as to effects of the mutation on rRNA processing, information will be obtained regarding the structural framework of processing, molecular interactions in processing, and the timing and order of these events. In addition, three rRNA processing events can be visualized co-transcriptionally, at low frequency but reproducibly. The three events visualized are thought to represent removal of the extreme 5' end of the transcript (the 5' ETS), separation of the bulk of the transcript from the extreme 3' end (the 3' ETS), and cleavage about midway through the transcript, which presumably corresponds to cleavage in ITS1 separating the 18S rRNA precursor from the rest of the precursor. These predictions will be tested using mutations in known required components, and if correct, effects on these specific processing steps will be tested in a structural setting. Components to be tested include both RNA and protein components of the two major classes of snoRNPs involved in rRNA modification (Box H/ACA and Box C+D), with an emphasis on the snoRNPs required for the early RNA cleavage steps, as well as non-snoRNP proteins involved in these steps. Completion of these experiments will provide important new information on early steps in ribosome biogenesis.