Ribosomes are ribonucleoprotein particles that contain 50-80 different proteins and 3-4 RNAs. These complex machines catalyze protein synthesis in almost all cells in nature. The long-term goal of this project is to understand how eukaryotic ribosomes are assembled in vivo. We use the yeast Saccharomyces cerevisae, to facilitate molecular genetic, biochemical, and proteomic approaches. Production of ribosomes is tightly linked to cell growth and proliferation. Consequently dysregulation of ribosome biogenesis is linked to many diseases such as cancer or developmental disorders. Because pathways of ribosome biogenesis are very conserved, our studies in yeast will help understand mechanisms of regulation and dysregulation of ribosome production in humans. In addition to ribosomal proteins and rRNAs, more than 200 assembly factors are present in nascent eukaryotic ribosomes and are required for their assembly. Production of these complex ribonucleoprotein machines requires a dynamic series of remodeling steps in which protein-protein, protein-RNA, and RNA-RNA interactions are established and reconfigured to produce functional ribosomes. Because most assembly factors are predicted to play a structural rather than enzymatic role, we need to understand in more detail how the networks of interactions among them and with r proteins and pre-rRNAs are established and reorganized to drive assembly. Potential conformational switches of RNA-RNA interactions that are likely to play an important role to regulate the timing and fidelity of assembly also need to be explored in more detail. We focus on twelve assembly factors and nearby ribosomal proteins that exhibit physical and functional interactions with each other and with pre-rRNA in precursors to 60S ribosomal subunits. We want to understand how this interaction network enables formation of stable precursor particles able to undergo processing of 27S precursors to 5.8S and 25S ribosomal RNAs. We will use genetic selections and site-directed mutagenesis to generate mutations in these proteins or pre-rRNAs, designed to disrupt interactions, including potential conformational switches in pre-rRNAs. We will then employ a battery of assays, and adapt new ones, to investigate effects of these mutations on pre-rRNA folding and processing, and assembly of ribosomal proteins into preribosomes.
This proposal describes research to study how ribosomal RNA, ribosomal proteins, and ribosome assembly factors interact with each other to drive assembly of ribosomes in yeast. As the ribonucleoprotein machines that catalyze protein synthesis in all cells, ribosomes are essential for and closely linked to the growth, proliferation, and adaptation of cells. Consequently dysregulation of ribosome synthesis results in cancers, anemias, mental retardation, and a variety of developmental disorders.
|Talkish, Jason; Campbell, Ian Winsten; Sahasranaman, Aarti et al. (2014) Ribosome assembly factors Pwp1 and Nop12 are important for folding of 5.8S rRNA during ribosome biogenesis in Saccharomyces cerevisiae. Mol Cell Biol 34:1863-77|
|Talkish, Jason; May, Gemma; Lin, Yizhu et al. (2014) Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA 20:713-20|
|Woolford Jr, John L; Baserga, Susan J (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195:643-81|
|Dembowski, Jill A; Ramesh, Madhumitha; McManus, C Joel et al. (2013) Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae. RNA 19:1639-47|
|Dembowski, Jill A; Kuo, Benjamin; Woolford Jr, John L (2013) Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits. Nucleic Acids Res 41:7889-904|
|Shimoji, Kaori; Jakovljevic, Jelena; Tsuchihashi, Kanako et al. (2012) Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. Nucleic Acids Res 40:4574-88|
|Bartoli, Kristen M; Jakovljevic, Jelena; Woolford Jr, John L et al. (2011) Kinesin molecular motor Eg5 functions during polypeptide synthesis. Mol Biol Cell 22:3420-30|
|Staley, Jonathan P; Woolford Jr, John L (2009) Assembly of ribosomes and spliceosomes: complex ribonucleoprotein machines. Curr Opin Cell Biol 21:109-18|
|Tang, Lan; Sahasranaman, Aarti; Jakovljevic, Jelena et al. (2008) Interactions among Ytm1, Erb1, and Nop7 required for assembly of the Nop7-subcomplex in yeast preribosomes. Mol Biol Cell 19:2844-56|
|Zhang, Jingyu; Harnpicharnchai, Piyanun; Jakovljevic, Jelena et al. (2007) Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes. Genes Dev 21:2580-92|
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