The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.The formation of eukaryotic ribosomes involves the production and correct assembly of four rRNAs and Ϸ80 ribosomal proteins. Due to its amenability for genetic and proteomic analyses, Saccharomyces cerevisiae is the organism where the different steps of this pathway have been best characterized (4,11,13,18,32,34). Thus, it is known that three of the four mature rRNAs that form the ribosome structure are generated from a common 35S pre-rRNA polycystronic precursor. After being transcribed in the nucleolus, this precursor is chemically modified and cleaved at three positions (known as the A 0 , A 1 , and A 2 sites) of its 5Ј-terminal end to generate the intermediate 33S, 32S, 27SA 2 , and 20S pre-rRNA precursors (see Fig. S1 in the supplemental material). The 20S and 27SA 2 pre-rRNAs then follow two independent maturation routes that lead to the generation of either the 18S rRNA (a component of the 40S ribosomal subunit) or the 5.8S and 25S rRNAs (two components of the 60S ribosomal subunit), respectively (see Fig. S1 in the supplemental material). These pre-rRNA maturation steps require the involvement of Ϸ170 nonribosomal proteins and 70 small nucleolar ribonucleoproteins (snoRNPs) (4,11,13,32). Different subsets of these molecules form large ribonucleoprotein complexes with specific pre-rRNA precursors that, according to their specific Svedberg coefficients in gradient ultracentrifugation experiments, were initially referred to as 90S, 66S, and 43S preribosomal particles (31, 33). The 90S particle, also known as the "small-subunit processome," contains the 35S pre-rRNA and assembly/processing factors needed for the early cleavage of the 35S pre-rRNA precursor at A 0 , A 1 , and A 2 sites, which is strictly required for the production of 40S ribosomal subunits. The 66S and 43S pa...

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.