Eukaryotic ribosome biogenesis is emerging as an unexplored target for cancer treatment. The tumor- suppressor proteins Rb, Art and p53 simultaneously downregulate cell division and ribosome biogenesis;conversely the oncogenic proteins c-Myc, Ras and PI3K upregulate both processes. Upregulation of ribosome biogenesis ensures that reaching critical cell mass does not limit cell proliferation. Importantly, the effective chemotherapeutic agent 5-fluorouracil inhibits processing of the precursor ribosomal RNA (pre- rRNA), an essential step of ribosome biogenesis and hence of cellular growth. The long-term objective is to decipher how essential proteins and their assemblies direct the dynamic RNA rearrangements that occur during a key step of ribosome biogenesis;namely hybridization between two sites of the U3 small nucleolar RNA (snoRNA) and two complementary sites in the pre-rRNA. This work will fill a major knowledge gap and is a prerequisite for therapies designed to inhibit proliferation of cancer cells by impairing a key biogenesis step. A barrier to achieving this objective is a lack of appropriate biochemical assays. To surmount this barrier, the PI has developed an in vitro assay to monitor formation of the U3-pre-rRNA duplexes and determined that the proteins ImpSp and Imp4p mediate formation of these duplexes. The central hypotheses are that a ternary complex between ImpSp, Imp4p and the U3 snoRNA lowers hybridization barriers (e.g. duplex instability or a buried base pairing site) to stimulate formation of the U3-pre-rRNA duplexes and that assembly of these proteins with their interacting partner, MpplOp, modulates activities of the individual proteins. To provide the thermodynamic and kinetic framework needed to address these hypotheses the PI has developed several assays based on fluorescence resonance energy transfer. The assays developed by the PI place his laboratory in a unique position to advance understanding of ribosome biogenesis and test these hypotheses by pursuing the four aims: probe the molecular mechanism by which the ternary complex lowers the (1) thermodynamic barrier to stability of the U3-ETS duplex and (2) kinetic barrier to formation of the U3-18S duplex;(3) describe how assembly of the ternary complex and addition of the interacting partner protein MpplOp modulates the site-specific annealing activities;and (4) determine the structural basis of the RNA binding and annealing activity and assembly of the ternary complex by X-ray crystallography.
|Plantinga, Matthew J; Korennykh, Alexei V; Piccirilli, Joseph A et al. (2011) The ribotoxin restrictocin recognizes its RNA substrate by selective engagement of active site residues. Biochemistry 50:3004-13|
|Gérczei, Tímea; Shah, Binal N; Manzo, Anthony J et al. (2009) RNA chaperones stimulate formation and yield of the U3 snoRNA-Pre-rRNA duplexes needed for eukaryotic ribosome biogenesis. J Mol Biol 390:991-1006|