Biogenesis of the Small Ribosomal Subunit in Eukaryotes

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DESCRIPTION (provided by applicant): All cells require ribosomes for translation, the process of decoding messenger RNA and synthesizing proteins. To satisfy the translational needs of a rapidly dividing eukaryotic cell, thousands of ribosomes must be synthesized per minute in a process that consumes a large portion of the cell's energy budget. Indeed, the rapid growth of many cancers requires up-regulation of ribosome biogenesis. Thus, understanding the mechanisms regulating ribosome biogenesis will provide insight for the development of new tools for controlling cell proliferationin disease states. The delineation of fundamental cellular pathways such as ribosome biogenesis is also critical for building the intellectual foundation for translational research. Many of the fundamental steps of eukaryotic ribosome biogenesis are poorly understood. Our functional genetic approach (Johnson lab) combined with the development of in vitro biochemical models (Correll lab) places us in a particularly strong position to study a fundamental step in eukaryotic ribosome biogenesis; the transition from the pre-ribosome particle (90S) to the pre-40S particle (small subunit precursor). Our overarching model is that the methyltransferase Bud23 monitors that status of 40S assembly, triggering the RNA helicase Ecm16 to promote release of the pre-40S from 90S only after completion of transcription and critical folding of the RNA. A major achievement in our preliminary results is our ability to trap an Ecm16-intermediate particle. To begin to address this model, our studies test four specific hypotheses: (i) Ecm16 is the helicase that dissociates U3 snoRNA and its associated proteins from the pre-rRNA (ii) Bud23 activates this helicase activity; (iii) Imp4 stabilizes the duplex that is the target of Ecm16; and (iv) the ribosomal protein Rps2 chaperones formation of the central pseudoknot, a key structural feature in 18S rRNA whose formation is sterically blocked until Ecm16 disrupts the U3-pre-rRNA interactions. The pre-rRNA processing pathways and the genes to be studied in the yeast Saccharomyces cerevisiae have counterparts in higher eukaryotes. Hence, S. cerevisiae is our model organism of choice because it allows us to combine powerful molecular genetic and biochemical approaches.