Eukaryotic Ribosome Assembly

Project Summary Ribosomes are responsible for the rapid and accurate production of all proteins in cells in all forms of life on earth. The ability of these molecular machines to carry out faithful translation depends on their complex structure that allows dynamic interaction with ligands. The mature ribosome in eukaryotic cells is composed of two parts, the large 60S subunit that synthesizes all the proteins in a cell and the small 40S subunit that decodes mRNA. The production of a eukaryotic ribosome involves over 200 accessory factors which orchestrate the intricate processing and folding of the ribosomal RNA and assembly of the ribosomal proteins. Considering the complexity of ribosome structure and function and its critical role in decoding our genetic information, ensuring their correct assembly is a necessary but daunting task for cells. Lately, considerable interest has been focused on mechanisms of quality control in the ribosome biogenesis pathway. This proposal focuses on two distinct topics within ribosome assembly; (1) the mechanisms for assessing the structural and functional integrity of the newly assembled 60S subunit and (2) the transition from the early 90S pre-ribosomal precursor to the pre-40S precursor. With respect to assessment of structural and functional integrity, this proposal addresses three related questions: I. How are newly minted subunits assessed for function? II. What is the fate of defective subunits? III. What is the consequence of licensing defective ribosomes? In humans, defects in these processes lead to various diseases, including T-cell acute lymphoblastic leukemia and Shwachman-Diamond syndrome. Assembly of the small ribosomal subunit involves stepwise cotranscriptional assembly of the 90S particle, a large protein-RNA complex, scaffolded on U3-snoRNA. However, the presence of U3 is mutually incompatible with the final folded structure of small subunit RNA and must be removed once transcription of the RNA is complete and the 90S particle has fully assembled. The transition from the 90S to pre-40S is poorly understood. We propose that the displacement of U3 by the RNA helicase Dhr1 is a primary event that drives the transition of the 90S into the pre-40S particle. We will determine how the activity of Dhr1 is regulated to ensure the timely release of U3.