Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (.5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. TABLE OF CONTENTS Abstract 643Introduction 644
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 (...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.