Ultrastructural Analysis of RNA Synthesis and Processing

该项目解决了真核核糖体如何在活细胞中组装的长期问题。这一过程既快速又极其复杂，需要200多种蛋白质和多种RNA。这些成分共同作用来修饰、切割和折叠核糖体rna，并确保核糖体蛋白的适当沉积，尽管人们对这一过程知之甚少。拜尔实验室采用一种独特的方法来研究核糖体组装。具体来说，在活性rRNA基因的背景下，通过电子显微镜（Miller染色质扩散）可以看到新生核糖体RNA转录本。这些基因编码大的前RNAs，这些前RNAs被切割产生主要的小亚基rna和大亚基rna。对一个活性基因上显示的多个转录本上形成的结构进行分析，每个转录本都比邻近的转录本年长或年轻几秒钟，从而可以阐明非常快速和非常动态的组装过程。使用这种方法，在小核糖体亚基组装中发现了多个离散的组装步骤，从而可以假设单个中间体的RNP组成以及特定重组步骤所需的组件。本项目的目标是通过对特定RNA和蛋白质组分的探针来验证这些假设，从而测试特定必需snoRNA（包括U3和U14 snoRNA）和特定必需蛋白的存在、位置和沉积时间。该项目涉及开发用于探测米勒染色质扩散中的特定rna和蛋白质的改进方法。这些实验的成功完成将大大有助于理解真核核糖体组装的共同目标。更广泛的影响：该项目将通过PI实验室与UVA暑期研究实习计划（SRIP）之间的安排，扩大代表性不足的少数民族学生在科学领域的代表性，该计划每年招募一名本科生到该实验室实习。计划中的研究非常适合学生的研究项目，也非常适合整合到PI关于转录和RNA加工的课堂教学中。结果将通过出版物和会议发言广泛传播。这项研究还有望产生更广泛的影响，为真核核糖体组装极其复杂的过程提供一个视觉窗口和新的见解，这将补充许多其他实验室正在采用的生化，遗传和蛋白质组学方法。

This project addresses the long-standing question of how a eukaryotic ribosome is assembled in a living cell. The process is both very rapid and extremely complex, requiring over 200 proteins and multiple RNA species. These components work together to modify, cleave and fold the ribosomal RNAs and ensure proper deposition of ribosomal proteins, though the process is poorly understood. The Beyer laboratory takes a unique approach to study ribosome assembly. Specifically, nascent ribosomal RNA transcripts are visualized by electron microscopy (Miller chromatin spreading) in the context of an active rRNA gene. These genes encode large pre-rRNAs that are cleaved to yield the major small and large subunit RNAs. Analysis of the structures forming on the multiple transcripts displayed on an active gene, each one a few seconds older or younger than its neighboring transcripts, allows elucidation of the very rapid and very dynamic assembly process. Using this method, multiple discrete assembly steps have been discovered in small ribosomal subunit assembly, allowing hypotheses about the RNP composition of the individual intermediates and the required components for specific restructuring steps. The goal of this project is to test these hypotheses using probes to specific RNA and protein components, thus testing the presence, location, and deposition time of specific essential snoRNAs (including U3 and U14 snoRNA) and specific essential proteins. The project involves the development of improved methods for probing specific RNAs and proteins in Miller chromatin spreads. Successful completion of these experiments will significantly contribute to the common goal of understanding eukaryotic ribosome assembly.Broader impact: This project will broaden the representation of under-represented minority students in science via an arrangement between the PI's lab and the Summer Research Internship Program (SRIP) at UVA, in which one undergraduate student is recruited annually to the program for placement in this lab. The planned research is well suited for student research projects and is also well suited for integration into the PI's classroom teaching on transcription and RNA processing. Results will be disseminated broadly via publications and meeting presentations. The research also promises to have the broader impact of providing a visual window and new insight into the extremely complex process of eukaryotic ribosome assembly, which will complement the biochemical, genetic and proteomic approaches being taken by many other laboratories.