In eukaryotic cells the final maturation of ribosomes occurs in the cytoplasm, where trans-acting factors are removed and critical ribosomal proteins are added for functionality. Here, we have carried out a comprehensive analysis of cytoplasmic maturation, ordering the known steps into a coherent pathway. Maturation is initiated by the ATPase Drg1. Downstream, assembly of the ribosome stalk is essential for the release of Tif6. The stalk recruits GTPases during translation. Because the GTPase Efl1, which is required for the release of Tif6, resembles the translation elongation factor eEF2, we suggest that assembly of the stalk recruits Efl1, triggering a step in 60S biogenesis that mimics aspects of translocation. Efl1 could thereby provide a mechanism to functionally check the nascent subunit. Finally, the release of Tif6 is a prerequisite for the release of the nuclear export adaptor Nmd3. Establishing this pathway provides an important conceptual framework for understanding ribosome maturation.

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

Eukaryotic ribosomes are preassembled in the nucleus and mature in the cytoplasm. Release of the antiassociation factor Tif6 by the translocase-like guanosine triphosphatase Efl1 is a critical late maturation step. In this paper, we show that a loop of Rpl10 that embraces the P-site transfer ribonucleic acid was required for release of Tif6, 90 Å away. Mutations in this P-site loop blocked 60S maturation but were suppressed by mutations in Tif6 or Efl1. Molecular dynamics simulations of the mutant Efl1 proteins suggest that they promote a conformation change in Efl1 equivalent to changes that elongation factor G and eEF2 undergo during translocation. These results identify molecular signaling from the P-site to Tif6 via Efl1, suggesting that the integrity of the P-site is interrogated during maturation. We propose that Efl1 promotes a functional check of the integrity of the 60S subunit before its first round of translation.

Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell-autonomous and non-cell-autonomous growth control roles of NS3 in Drosophila and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.