Susan Baserga, MD, PhD

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.

Specialized Terms: Ribosome biogenesis; RRNA processing; U3 RNP structure and function; RNA helicases; Polymerase I transcription and processing

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

identifying
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.