The long-term goal of the proposed research is to better understand the molecular mechanisms that regulate RNA polymerase I transcription of ribosomal RNA. This rRNA becomes the major structural and functional component of ribosomes and its rate of synthesis determines the rate of ribosome biogenesis, which in turn is directly correlated with cell growth rate. Thus it is of great importance to understand this process so as to understand the increased growth characteristic of cancer cells. Indeed, many cancer therapeutics that target rapidly growing cells function in part by decreasing RNA polymerase I transcription and new drugs are being tested that specifically target this process. Huge advances have been made in structural and functional analyses of the polymerase itself and of additional factors that participate in making ribosomal RNA, yet many important questions remain unanswered. The Beyer laboratory contributes a unique in vivo approach to this research effort by using electron microscopy to directly visualize active rRNA genes released from growing cells. The Miller chromatin spreading method allows high-resolution analysis of many individual genes as well as analysis of the gene population in many individual nucleoli. Using Saccharomyces cerevisiae as a model system, rRNA genes are visualized in the presence and absence of key regulatory proteins (i.e., in appropriate mutant strains) to determine how these proteins affect rRNA synthesis at the individual gene level in living cells. Changes or defects in transcription initiation, elongation, termination, processing of nascent transcripts, chromatin structure and template topology can be discerned and distinguished, as well as any coupling between these processes. Quantitative analysis of many individual genes allows determination of statistically significant changes and provides mechanistic insight, such as distinction between elongation factors that contribute to recognition of intrinsic pause sites and those that help polymerases transit such sites. The specific goals of this project are (1) to characterize the in vivo function of RNA polymerase I subunits in rRNA transcription and transcription-coupled RNA processing, (2) to characterize the in vivo function of two elongation factors, Spt5 and FACT, in rRNA transcription and transcription-coupled RNA processing, and (3) to investigate transcription termination by RNA polymerase I and identify contributions from nascent transcript degradation, intrinsic pause sites, protein roadblocks and RNA cleavage activity intrinsic to the polymerase. The information gained by direct visualization of active genes from living cells is extremely valuable when combined with genetic and biochemical analyses from other investigators, and together provide a much more complete picture of the regulation of ribosomal RNA synthesis by RNA polymerase I.

Public Health Relevance

The goal of this project is to help determine how cells control the production of ribosomes, which are the cell's protein-synthesizing machines and thus are required for rapid cell growth and are increased in cancer cells. The project focuses on the first step in the process - making large ribosomal RNAs - because this step controls the rate at which ribosomes are made. Information gained by a unique approach that involves direct visualization of active genes will reveal regulatory steps that may be promising new targets in cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063952-11
Application #
8293224
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Sledjeski, Darren D
Project Start
2001-08-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
11
Fiscal Year
2012
Total Cost
$320,166
Indirect Cost
$112,266
Name
University of Virginia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Harris, Bethany; Bose, Tania; Lee, Kenneth K et al. (2014) Cohesion promotes nucleolar structure and function. Mol Biol Cell 25:337-46
Axt, Konstantin; French, Sarah L; Beyer, Ann L et al. (2014) Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage. PLoS One 9:e85703
Zhang, Yinfeng; Anderson, Susan J; French, Sarah L et al. (2013) The SWI/SNF chromatin remodeling complex influences transcription by RNA polymerase I in Saccharomyces cerevisiae. PLoS One 8:e56793
Viktorovskaya, Olga V; Engel, Krysta L; French, Sarah L et al. (2013) Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II. Cell Rep 4:974-84
Johnson, Joseph M; French, Sarah L; Osheim, Yvonne N et al. (2013) Rpd3- and spt16-mediated nucleosome assembly and transcriptional regulation on yeast ribosomal DNA genes. Mol Cell Biol 33:2748-59
French, Sarah L; Sikes, Martha L; Hontz, Robert D et al. (2011) Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes. Mol Cell Biol 31:482-94
Anderson, Susan J; Sikes, Martha L; Zhang, Yinfeng et al. (2011) The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively. J Biol Chem 286:18816-24
Huber, Alexandre; French, Sarah L; Tekotte, Hille et al. (2011) Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L. EMBO J 30:3052-64
El Hage, Aziz; French, Sarah L; Beyer, Ann L et al. (2010) Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev 24:1546-58
Zhang, Yinfeng; Sikes, Martha L; Beyer, Ann L et al. (2009) The Paf1 complex is required for efficient transcription elongation by RNA polymerase I. Proc Natl Acad Sci U S A 106:2153-8

Showing the most recent 10 out of 25 publications