ReviewRibosome assembly in eukaryotes
Introduction
In eukaryotes, ribosome biogenesis takes place in the nucleolus, a specialized compartment within the nucleus. The process starts with the synthesis of 5S and 35S pre-rRNAs by distinct RNA polymerases and requires the import of ribosomal proteins from the cytoplasm. The mature RNA components (5.8S, 25S/28S and 5S rRNA for the 60S subunit and 18S for the 40S subunit), are released from pre-rRNAs following a complex pathway that involves both endo- and exonucleolytic digestions. Concomitantly, pre-rRNAs are extensively modified and bound by the ribosomal proteins before the assembled pre-40S and pre-60S subunits are exported separately to the cytoplasm (reviewed in Venema and Tollervey, 1999). The various rRNAs and ribosomal proteins are produced in equimolar amounts and their synthesis is tightly regulated by a variety of growth conditions (reviewed in Woolford and Warner, 1991).
Maturation of the ribosomal rRNA and its assembly into ribosomal subunits involves at least 170 accessory proteins comprising endo- and exoribonucleases, putative ATP-dependent RNA helicases, ‘chaperones’ or ‘assembly factors’ (Venema and Tollervey, 1999, Kressler et al., 1999a) and about as many small nucleolar ribonucleoprotein particles (snoRNPs). This large number is related to the peculiar and elaborate system of ribose methylation or pseudouridine formation in eukaryotes where the specificity of the target site is dictated by RNA hybridization between the small nucleolar RNAs (snoRNAs) and the pre-rRNA (Ofengand and Bachellerie, 1998, Bachellerie et al., 2000). At least four snoRNPs, including the U3 snoRNP, participate in the early cleavages of the primary 7 kb 35S transcript. The 35S forms with non-ribosomal and ribosomal proteins a large RNP complex that is rapidly converted into precursors of the 40S and 60S ribosomal subunits. The pre-40S particles are further processed in the cytoplasm, whereas the maturation of the pre-60S continues in the nucleus before export to the cytoplasm. Therefore, formation of eukaryotic ribosomes not only requires the coordination of processing and assembly events but also a spatio-temporal ordering of these steps starting in the nucleolus and ending in the cytoplasm. This raises the question of how the different trans-acting factors determine the correctness of the assembly process or the export competence of the pre-ribosomal particles across the nuclear pores.
From in vitro reconstitution assays made with bacterial systems, we learned that assembly of the ribosomal subunits from the individual proteins and rRNA components is a self-assembly process that only requires a heating step (reviewed in Nierhaus, 1991). In vivo, additional factors that decrease the activation energy of rate-limiting reactions either in the complex folding of the rRNA or in the rearrangement of the RNA-proteins interactions are needed. It is not surprising that only a few ribosome accessory proteins hydrolyzing ATP have been discovered in Escherichia coli. These include RNA helicases (Nierhaus, 1991) and more recently the DnaK/hsp 70 chaperone (Maki et al., 2002; reviewed in Woolford, 2002). In eukaryotes, the situation is more complicated and the rRNA maturation requires a large number of pre-ribosomal factors.
Until recently, the identification of many processing and assembly factors in eukaryotes was made on the basis of biochemical and genetic studies mostly done in yeast. Some of them were first characterized biochemically in higher eukaryotic cells and because of the existence of yeast homologues further analyzed in Saccharomyces cerevisiae. This led to rapid advances in understanding ribosome synthesis and indicated that the principles that govern ribosome assembly in yeast apply to eukaryotes in general. A limited network of interactions among factors could also be established using synthetic lethal screens or high-copy suppression in yeast. Recently, the use of proteomic approaches (Gavin et al., 2002, Grandi et al., 2002, Nissan et al., 2002) to identify the set of proteins and RNA components associated with epitope-tagged factors involved in ribosome biogenesis (Bassler et al., 2001, Harnpicharnchai et al., 2001, Saveanu et al., 2001) opens novel perspectives in correlating the protein composition of the nascent pre-ribosomes with a given state of the rRNA processing. Several distinct intermediates have already been characterized and this strategy seems the most promising in generating a coherent picture for the RNA processing and ribosome assembly pathways in eukaryotes.
Section snippets
The RNA processing pathway
The formation of functional ribosomes in yeast starts with the synthesis of pre-rRNAs by the activity of the RNA polymerase I. The first detectable intermediate (35S in yeast) contains 5′ and 3′ external transcribed region (ETS) as well as the mature 18S rRNA, 5.8S and 25S rRNA interspersed with non coding sequences ITS1 and ITS2. The 18S rRNA will be the rRNA component of the small 40S subunit whereas the 5.8S and 25S rRNA together with the 5S rRNA, synthesized independently by RNA polymerase
rRNA modifications and the factors involved
The isomerization of uridines to pseudouridines (Ψs) and methylation of 2′-hydroxyl of riboses are the most prevalent modified nucleotides in rRNAs. About 100 rRNA sites of each type are modified in human (Maden, 1990). This number decreases to 50 in the yeast Saccharomyces cerevisiae and E. coli ribosomes contain only four ribose-methylated nucleotides and ten pseudouridines in addition to ten base methylations at various positions (Rozenski et al., 1999). Two main features are associated with
Ribosome assembly
Eukaryotic ribosome assembly is best understood in yeast where a large number of ribosome precursors have been isolated and their components identified. The non-ribosomal proteins associated with pre-ribosomal particles are for a large majority of them essential for ribosome biogenesis. Other factors, discovered by genetic screens, are involved in ribosome assembly although their stable association with intermediate particles remains unclear. In total, and excluding the ribosomal proteins,
The nucleolar ribosome synthesis
The organization of the nucleolus into three substructures, the fibrillar centers (FCs), the dense fibrillar compartment (DFC) and the granular component (GC) is now well established for lower and higher eukaryotes. By sequestering specific ribosome processing factors, these ultrastructures are believed to participate in the spatio-temporal ordering of the ribosome synthesis pathway (Scheer and Hock, 1999). The analysis of the DFC revealed that it contains fibrillarin (Nop1p in yeast), snoRNAs
Intranuclear pre-60S particles assembly and transport
Based on the different intracellular or intranuclear distribution of pre-ribosomal proteins detected by immunofluorescence or electron microscopy as well as on the different protein composition of various pre-60S particles, a number of successive pre-60S complexes can be defined, as discussed in the sections 4.5 and 4.6.
It is admitted that pre-60S processing/assembly takes place essentially in the nucleolus, with a short, transient, nucleoplasmic stage followed by export to the cytoplasm. Many
Regulation of ribosome biogenesis at the level of nucleocytoplasmic exchanges
A characteristic feature of all eukaryotic cells is the existence of a lipidic bilayer surrounding the genetic material therefore separating the site of transcription from that of translation. This implies that free exchanges of macromolecules between the nucleus and the cytoplasm are not allowed and have to take place through nuclear pore complexes (NPCs) which act as a discriminating gate (Rout and Aitchison, 2001). While small molecules can diffuse rapidly and efficiently through NPCs,
Nuclear ATPases and GTPases at work during ribosome assembly
Formation of ribosomes is a dynamic process involving a large number of intermediates. Many steps in this complex pathway involve structural transitions, which probably require an input of energy. It is thus not surprising that ‘energy-rich molecules’ hydrolyzing either ATP or GTP are involved at various stages of the assembly process.
A large class of ATPases consists of putative ATP-dependent RNA helicases of the DExH/D box family which may use their RNA unwinding activity to allow RNA/RNA
Lessons from the analysis of the recent 3D structures and implication of ‘RNP’ chaperones
Ribosomal proteins play an active role in the assembly process. In vitro reconstitutions of active ribosomes with bacterial systems have focused on two important principles of the assembly process: binding of the ribosomal proteins is ordered and cooperative and completion of the process is temperature dependent (reviewed in Nomura, 1990, Nierhaus, 1991).
Details of the structures of individual ribosomal proteins and their interactions with RNA have been analyzed in the context of the 30S
Conclusions and perspectives
Ribosome biogenesis in eukaryotes involves many pre-ribosomal complexes that we begin to understand in terms of their intracellular localization, pre-rRNA and protein composition. The identification of the associated proteins revealed the possible function of many proteins of previously uncharacterized function. We learned that the maturation of the pre-60S particles takes place not only in the nucleolus but also in the nucleoplasm and the cytoplasm. A surprising discovery was that the majority
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