Therapeutic Radiology/Radiation Oncology: Radiobiology
Of Yeast and Ribosome Biogenesis: Ribosome biogenesis is a complex process requiring the coordinated expression of rRNA and protein moieties and their assembly in the eukaryotic nucleolus. In order to better understand each aspect of this process, we are using an array of genetic, biochemical, and cell biological techniques in the yeast Saccharomyces cerevisiae. My laboratory focuses on the role of the ribonucleoprotein and protein complexes involved in generating the mature rRNAs.
Extensive Research Description
Study of RNA helicases required for ribosome biogenesis and their cofactors
investigations into the role of ribosome biogenesis in cell cycle regulation
discovery of a subset of SSU processome proteins that are associated with the rDNA and are required for rDNA transcription
subcomplexes of the SSU processome and deciphering the direct
protein-protein and protein-RNA interactions that mediate their assembly
purification and electron microscopy to visualize pre-ribosomes
characterization of an essential new protein-protein interaction motif found in RNA processing RNPs
developing a method to identify individual proteins in chromatin spreads.
Using innovative proteomics techniques, my laboratory has recently identified the protein components of a large nucleolar ribonucleoprotein that is required for processing of the 18S small subunit rRNA. This RNP, which we termed the SSU processome, is composed of the U3 snoRNA and 40 proteins. Currently, projects in the lab are aimed at determining the architecture of this RNP and the functions of individual proteins in 18S processing. We approach this question from several perspectives, using genetic and biochemical methods to identify direct interactions between components, and cryo electron microscopy to visualize the complex in three dimensions.
Through these studies we have discovered and characterized several unique protein motifs and their specific roles in rRNA processing. We have recently discovered that a subset of the SSU processome proteins are associated with the rDNA and are required for rDNA transcription. Stemming from this idea, we are interested in studying the proteins which regulate transcription of the rDNA by Pol I and initiate the processing of the rRNA. We have learned that these steps are intimately linked, and endeavor to describe this complex process in detail. Seventeen putative RNA helicases have been shown to be required for processing of the small and large ribosomal subunit RNAs, perhaps by remodeling the rRNA to allow access to cleavage sites. Ongoing genetic and biochemical studies in the lab examine the roles of each putative RNA helicase and test its ability to unwind RNA. Through these projects, we strive to ascertain how and why the helicases are required at each step in ribosome biogenesis. Because ribosomes are essential to cell growth via the production of new proteins, we are studying the role of ribosome biogenesis in cell cycle regulation.
We have previously shown that rRNA maturation by the SSU processome is required for cell cycle progression, indicating that the production of ribosomes has a distinct influence on the cell cycle. Specifically, we seek to find the ribosome-regulated trigger that allows the cell to progress through the cell cycle, grow in size, and divide. Transcription of the rDNA and processing of the rRNA can be visualized in Miller chromatin spreads, as shown here. In a, the SSU processome corresponds to the terminal knobs at the end of each rRNA branching off the rDNA. When components of the SSU processome are depleted (the U3 snoRNA in b, or the Utp7 protein in c), the knobs are no longer present, due to incomplete formation of the SSU processome.
- Champion EA, Kundrat L, Regan L, Baserga SJ. A structural model for the HAT domain of Utp6 incorporating bioinformatics and genetics. Protein Eng Des Sel. 2009 Jul22(7):431-9. Epub 2009 Jun 10.
- A direct interaction between the Utp6 half-a-tetratricopeptide repeat domain and a specific peptide in Utp21 is essential for efficient pre-rRNA processing. Champion EA, Lane BH, Jackrel ME, Regan L, Baserga SJ. Mol Cell Biol. 2008 Nov28(21):6547-56. Ep
- Bernstein K.A., Bleichert F., Bean J.M., Cross F.R. and Baserga S.J. (2007). Ribosome biogenesis is sensed at the Start cell cycle checkpoint. Mol Biol Cell. 18, 953-64.
- Bleichert, F. and Baserga, S.J. (2007). The long unwinding road of RNA helicases. Mol Cell. 27, 339-52. Review.
- Granneman S, Lin C, Champion EA, Nandineni MR, Zorca C, Baserga SJ. The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis. Nucleic Acids Res. 2006 Jun 1334(10):3189-99. Print 2006.