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.
