Nug1 is a potassium-stimulated GTPase affecting the association of early 60S assembly factors in ribosome biogenesis

Abstract

Ribosomes are complex macromolecular machineries responsible for protein synthesis (translation) in all living cells. In yeast, they are composed of four rRNA species assembled with 79 ribosomal proteins to form the small (40S) and the large (60S) subunit. To reach their final translation-competent form, they go through a complex, highly dynamic and coordinated process termed ribosome biogenesis. In eukaryotes, more than 180 transiently associating non-ribosomal factors (assembly factors) and 70 small nucleolar RNAs (snoRNAs) are involved in rRNA processing and modifications, as well as in the assembly of r-proteins (Henras et al., 2008; Lafontaine and Tollervey, 2001; Staley and Woolford, 2009). Several of the 60S ribosome biogenesis factors belong to the superfamily of GTPases, including Nug1. Nug1 is a circularly permuted GTPase and an essential trans-acting factor in ribosome biogenesis. It co-purifies with various nucleolar and nucleoplasmic pre-ribosomal particles and exhibits RNA-binding properties (Bassler et al., 2001; Bassler et al., 2006). However, several questions remained open regarding the exact role of Nug1 in ribosome biogenesis, including the regulation of its enzymatic GTPase activity, its binding site on the pre-ribosome, as well as a possible role in the recruitment and/or release of other 60S assembly factors. During my PhD studies, I performed a series of in vitro GTPase and nucleotide binding assays using the C. thermophilum (CtNug1) orthologue to address Nug1’s enzymatic activity. With these, I showed that CtNug1 exhibits a low intrinsic GTPase activity that can be stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. I’ve also generated a series of point mutations in the G-domain that specifically inhibit GTP hydrolysis or nucleotide binding. The orthologous mutations in the yeast Nug1 GTPase domain were subsequently tested for their effects on ribosome biogenesis. Early 60S assembly factors including Dbp10, Spb1, Nop2 and Mrt4 associated less with affinity purified pre-ribosomal particles, when the Nug1 nucleotide-binding mutant (D446N) was expressed or when Nug1 was depleted. Interestingly, no growth defects or biochemical differences in pre-ribosomal particle composition were observed for the catalytic (G339A) mutant, suggesting that the GTP hydrolysis is not essential for Nug1’s function. From the early assembly factors affected, only the essential RNA helicase Dbp10 was genetically linked to Nug1 (Bassler et al., 2001). In collaboration with Dr. Emma Thomson, we identified the binding sites of Nug1 and Dbp10 onto the pre-ribosome using the CRAC technique. Both proteins were found to bind in close proximity to each other on the interface of the 60S subunit at the PTC area. Further, in vitro binding assays confirmed a physical interaction between Nug1 and Dbp10. Together the findings from my PhD thesis show that Nug1 affects the dynamic interplay of assembly factors including those localizing to the PTC area (Dbp10, Sbp1, Nop2, Nsa2), as well as factors involved in the P-stalk formation (Mrt4, Yvh1, Rpp0, Rpl12). In this interplay, the Nug1 binds at the base of helix 89 and may act as a molecular GTPase switch that mediates the crosstalk between the maturation of PTC and the P-stalk, two distinct and essential hallmarks of the 60S subunit.