We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.

Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (~30 species) dissociate, and signi®cantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.

Many RNA nucleases and helicases participate in ribosome biogenesis, but how they cooperate with each other is largely unknown. Here we report that in vivo cleavage of the yeast pre-rRNA at site D, the 3-end of the 18 S rRNA, requires functional interactions between PIN (PilT N terminus) domain protein Nob1 and the DEAH box RNA helicase Prp43. Nob1 showed specific cleavage on a D-site substrate analogue in vitro, which was abolished by mutations in the Nob1 PIN domain or the RNA substrate. Genetic analyses linked Nob1 to the late pre-40 S-associated factor Ltv1, the RNA helicase Prp43, and its cofactor Pfa1. In strains lacking Ltv1, mutation of Prp43 or Pfa1 led to a striking accumulation of 20 S pre-rRNA in the cytoplasm due to inhibition of site D cleavage. This phenotype was suppressed by increased dosage of wildtype Nob1 but not by Nob1 variants mutated in the catalytic site. In ltv1/pfa1 mutants the 20 S pre-rRNA was susceptible to 3 to 5 degradation by the cytoplasmic exosome. This degraded into the 3 region of the 18 S rRNA, strongly indicating that the preribosomes are structurally defective.Eukaryotic ribosome formation is a complex process that requires coordination of rRNA synthesis and processing with the subsequent incorporation of the ribosomal (Rpl and Rps) proteins (reviewed in Refs. 1-5). In the yeast Saccharomyces cerevisiae, ribosome synthesis starts with the transcription of a 35 S pre-rRNA, which is the common precursor to the mature 18, 5.8, and 25 S rRNAs. This pre-rRNA is co-transcriptionally bound by ribosomal proteins as well as many non-ribosomal trans-acting factors to form a 90 S preribosomal particle. Next, this 90 S intermediate undergoes a series of early pre-rRNA processing and modification steps in the nucleolus before an endonucleolytic cleavage at site A 2 in the pre-rRNA separates the pathways for 60 and 40 S subunit assembly.Pre-60 S particles undergo further maturation steps in the nucleus, whereas pre-40 S particles contain few non-ribosomal proteins and are rapidly exported to the cytoplasm. The factors involved in 40 S export are largely unknown, but the non-essential pre-40 S factor Ltv1 was suggested to play a role in this process (6). Following nuclear export, the pre-40 S subunit undergoes cytoplasmic maturation, involving two major events. (i) 20 S pre-rRNA cleavage at site D generates the 3Ј-end of the mature 18 S rRNA, and (ii) structural reorganization forms the characteristic beak structure of the mature 40 S subunit, resulting in exposure of RNA helix 33 within the 18 S rRNA (7).The sites and the order of endonuclease and exonuclease processing events that convert the 35 S pre-rRNA into the mature rRNA species are well characterized in yeast (Fig. 3A) (reviewed in Refs. 4 and 8). However, although most or all of the exonucleases participating in rRNA processing are known, several predicted endonucleases remain to be identified. Two proteins, Fap7 and Nob1, were proposed to cleave site D (9 -11), but for neither was endonuclease activity shown. Fap7 is a putativ...

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

Ribosome synthesis involves dynamic association of ribosome biogenesis factors with evolving pre-ribosomal particles. Rio2 is an atypical protein kinase required for pre-40S subunit maturation. We report the crystal structure of eukaryotic Rio2 with bound ATP/Mg2+. Unexpectedly, the structure reveals a phosphoaspartate intermediate with ADP/Mg2+ in the active site, typically found in Na+-, K+- and Ca2+-ATPases. Consistent with this finding, ctRio2 exhibits a robust ATPase activity in vitro. In vivo, Rio2 docks on the ribosome with its active site occluded, and its flexible loop positioned to interact with the pre-40S subunit. Moreover, Rio2 catalytic activity is required for its dissociation from the ribosome, a necessary step in pre-40S maturation. We propose that phosphoryl transfer from ATP to Asp257 in Rio2’s active site and subsequent hydrolysis of the aspartylphosphate could be a trigger to power late cytoplasmic 40S subunit biogenesis.