The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.

The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly intermediates that contain WD40 protein-protein interaction motifs. Such proteins may play key roles in organizing other molecules necessary for ribosome biogenesis. Ytm1 is present in four consecutive 66S preribosomes containing 27SA 2 , 27SA 3 , 27SB, and 25.5S plus 7S pre-rRNAs plus ribosome assembly factors and ribosomal proteins. Ytm1 binds directly to Erb1 and is present in a heterotrimeric subcomplex together with Erb1 and Nop7, both within preribosomes and independently of preribosomes. However, Nop7 and Erb1 assemble into preribosomes prior to Ytm1. Mutations in the WD40 motifs of Ytm1 disrupt binding to Erb1, destabilize the heterotrimer, and delay pre-rRNA processing and nuclear export of preribosomes. Nevertheless, 66S preribosomes lacking Ytm1 remain otherwise intact.Biogenesis of eukaryotic ribosomes is a highly regulated and dynamic process that begins in the nucleolus with transcription of a precursor rRNA (pre-rRNA) that is rapidly packaged into the 90S ribonucleoprotein particle containing ribosomal proteins, nonribosomal proteins, and snoRNA-containing ribonucleoprotein particles (snoRNPs). The 90S pre-RNPs are converted into 43S and 66S ribosome assembly intermediates, which ultimately give rise to mature 40S and 60S ribosomal subunits (Fig. 1).Molecular genetic approaches in yeast identified more than 70 trans-acting factors required for ribosome assembly (12,14,46). Subsequent advances in proteomics enabled purification of pre-rRNPs from yeast and identification of an additional 80 assembly factors present in preribosomes, as well as most of those proteins previously discovered using genetic screens (3,7,11,17,20,21,24,26,37,38,41,(49)(50)(51). Metazoan homologues of most of the yeast ribosome assembly factors were discovered by proteomic analysis of purified nucleoli (2, 52).Among the assembly factors found in yeast preribosomes are 17 proteins containing WD40 motifs (14). These motifs function as protein-protein interaction domains (53). Therefore, such WD40-containing proteins may nucleate assembly of preribosomes by interacting sequentially or simultaneously with other assembly factors or ribosomal proteins. Previously, we identified the WD40 protein Ytm1 as a constituent of purified 66S pre-rRNPs and showed that depletion of Ytm1 results in a deficiency of 60S ribosomal subunits (21).In this study, we have further investigated the role of Ytm1 in ribosome biogenesis. Ytm1 is a constituent of multiple consecutive 66S preribosomes containing 27SA 2 , 27SA 3 , 27SB, 25.5S, and 7S pre-rRNAs plus a collection of ribosomal and nonribosomal proteins. Ytm1 is present in a heterotrimer with two other assembly factors, Nop7 and Erb1, both within 66S pre-rRNPs and as a subcomplex independent of preribosomes. Mutations in Ytm1 disrupt interactions between Ytm1 and Erb1, destabilize the heterotrimer, and significantly reduce association of these three proteins with 66S preribosomes. These 66S pre-rRNPs ...

The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA 3 to 27SB S pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA 3 and 27SB L pre-ribosomal RNAs.

To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the ‘B-factor’ proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes.

Background Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced.Methodology/Principal FindingsFor each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity.Conclusions/SignificanceAs expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.