Ultrastructural Analysis of RNA Synthesis and Processing

核糖体是将遗传密码转化为蛋白质的生物机器，它在体内的组装是生物学中一个长期存在的问题。在这个项目中，小核糖体亚基生物发生的早期步骤将通过酵母中新生核糖体RNA转录物的电镜可视化来研究。在新生转录本上，小亚基的五个可重复组装中间体是可识别的，从形成一个小的5'颗粒开始，最终将整个18S rRNA序列压实成一个大的末端突起（前小亚基），并通过RNA切割共转录释放。值得注意的是，中间组装步骤涉及18S RNA中RNP形成的三个扭结节点的形成，这些节点大约映射到18S rRNA的三个已知结构域，并且在将RNA的长度压缩成大末端旋钮的过程中相互坍塌。这些事件让人想起细菌核糖体重构研究的发现，其中表明小亚基的三个亚域（体、平台、头）是自主组装域。因此，将测试的假设是RNP形成的三个节点代表核糖体蛋白在18S RNA的三个结构域上的组装，这将是该组装途径在体内的第一次测试。将使用酵母遗传学和电子显微镜相结合的方法，通过消耗早期组装的核糖体蛋白和插入rRNA扩增序列，来测试对淋巴结大小或间距的预测影响，从而验证这一假设。利用抗体或细胞化学标记将特异性蛋白定位于EM结构中。该项目将有助于阐明活细胞中真核核糖体的生物发生过程，并代表了对这一极其重要和复杂的生物过程的新的实验处理。它还将为学生提供培训机会，包括为来自未被充分代表的少数群体的本科生提供年度暑期研究实习机会。

A long-standing question in biology concerns the in vivo assembly of ribosomes, which are the biological machines that translate the genetic code into proteins. In this project, early steps in biogenesis of the small ribosomal subunit will be studied by electron microscopic visualization of nascent ribosomal RNA transcripts in the yeast, Saccharomyces cerevisiae. Five reproducible assembly intermediates for the small subunit are discernible on nascent transcripts, beginning with formation of a small 5' particle and culminating in compaction of the entire 18S rRNA sequence into a large terminal knob (the pre-small subunit), which is released co-transcriptionally by RNA cleavage. Remarkably, an intermediate assembly step involves formation of three kinked nodes of RNP formation in the 18S RNA, which map approximately to the three known domains of 18S rRNA and which collapse onto each other during a process that dramatically compacts the length of the RNA into the large terminal knob. These events are reminiscent of findings from bacterial ribosome reconstitution studies, in which it was shown that the three subdomains of the small subunit (body, platform, head) are autonomous assembly domains. Thus, the hypothesis that will be tested is that the three nodes of RNP formation represent assembly of ribosomal proteins onto the three structural domains of 18S RNA, which will be the first test of this assembly pathway in vivo. A combination of yeast genetics and electron microscopy will be used to test the hypothesis by depletion of early-assembling ribosomal proteins and by insertion of rRNA expansion sequences to test for predicted effects on the size or spacing of nodes. Specific proteins will be localized in the structures using antibody or cytochemical labeling for EM. This project will help elucidate the process of eukaryotic ribosome biogenesis in living cells, and represents a new experimental handle on this extremely important and complex biological process. It will also present training opportunities for students, including an annual summer research internship for an undergraduate from an underrepresented minority group.