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

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

The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation

Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA–protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms

Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export

Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes (1-3). Nuclear export is a key, irreversible step in pre-ribosome maturation(4-8), but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved GTPase Nug2/Nog2 (called NGP-1, Gnl2 or nucleostemin 2 in human(9)) plays a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor(10). Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding prior to Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodeling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K(+)-dependent GTPase activity and the remodeling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery