Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm1,2. Hundreds of assembly factors, organized into sequential functional groups3,4, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.

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.

More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors-e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs-remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm. In eukaryotes, 79 ribosomal proteins associate with ribosomal RNA (rRNA) to produce 40S and 60S ribosomal subunits (Woolford and Warner 1991). Three of the four rRNAs in mature ribosomes are derived from the 35S-45S rRNA precursor (pre-rRNA) transcribed by RNA polymerase I, while the fourth rRNA, 5S rRNA, is transcribed from separate genes by RNA polymerase III. The 35S-45S primary transcript is packaged into a 90S ribonucleoprotein particle (RNP), together with a subset of assembly factors and ribosomal proteins. Subsequent steps trigger folding, modification, and processing of prerRNAs and association of additional assembly factors and ribosomal proteins in 43S and 66S assembly intermediates. These pre-rRNPs undergo further maturation in the nucleolus, nucleoplasm, and then cytoplasm to form functional 40S and 60S ribosomal subunits, respectively ( Fig. 1A; FromontRacine et al. 2003;Raué 2003;Granneman and Baserga 2004). Preribosomal particles in the assembly pathway are distinguished by the presence of successive prerRNA processing intermediates (Fig. 1A). However, it is not clear into which of the consecutive preribosomes 5S rRNA and each ribosomal protein are incorporated, which assembly factors are required to recruit these molecules, or how they do so. Furthermore, the mechanisms by which constituents of nascent ribosomes facilitate folding, processing, and modification of pre-rRNAs remain elusive.5S rRNA is essential for maturation of preribosomes and for the function of mature ribosomes (Van Ryk et al. 1992;Dechampesme et al. 1999;Kiparisov et al. 2005). Steitz and coworkers defined a pathway of assembly of 5S rRNA into ribosomes in HeLa cells. Newly synthesized 5S pre-rRNA binds transiently to the La protein (Rinke and Steitz 1982;Yoo and Wolin 1994). Following 3Ј-end maturation, 5S rRNA binds to ribosomal protein rpL5, then assembles into ribosomes (...

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 ...

Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with ribosomal RNA (rRNA) provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in precursor rRNA (pre-rRNA) processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To test this model, we used proteomic approaches to assay changes in the levels of ribosomal proteins and assembly factors in preribosomes when RPLs functioning in early, middle, and late steps of pre-60S assembly are depleted. Our results demonstrate that structural domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic 60S subunits. Our results reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into how these RNA-protein particles evolved.

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

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.

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA 3 to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA 3 pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A 3 assembly factors and for proper assembly of the neighborhoods containing domains I and II.