Ribosome biogenesis is a fundamental process that provides cells with the molecular factories for cellular protein production. Accordingly, its misregulation lies at the heart of several hereditary diseases (e.g., Diamond-Blackfan anemia). The process of ribosome assembly comprises the processing and folding of the pre-rRNA and its concomitant assembly with the ribosomal proteins. Eukaryotic ribosome biogenesis relies on a large number (>200) of non-ribosomal factors, which confer directionality and accuracy to this process. Many of these non-ribosomal factors fall into different families of energy-consuming enzymes, notably including ATP-dependent RNA helicases, AAA-ATPases, GTPases, and kinases. Ribosome biogenesis is highly conserved within eukaryotic organisms; however, due to the combination of powerful genetic and biochemical methods, it is best studied in the yeast Saccharomyces cerevisiae. This review summarizes our current knowledge on eukaryotic ribosome assembly, with particular focus on the molecular role of the involved energy-consuming enzymes.

SummaryEukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1’s meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.

2The synthesis of ribosomes is one of the major cellular activities, and in eukaryotes, it takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (125, 155). There, the rRNA genes are transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs is intimately linked to their assembly with the ribosomal proteins (r-proteins). These processes depend on various cis-acting elements (6, 188), and they require a large number of nonribosomal protein trans-acting factors (97,174,193). Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetic and biochemical approaches in the yeast Saccharomyces cerevisiae. This minireview is aimed at giving an insight into the functions of the many protein trans-acting factors involved in ribosome biogenesis in S. cerevisiae. PRE-rRNA PROCESSING AND RIBOSOME ASSEMBLY PATHWAYSIn S. cerevisiae, the large 60S ribosomal subunits are composed of 46 r-proteins and three rRNA species (5S, 5.8S, and 25S) while the small 40S ribosomal subunits contain 32 rproteins and the 18S rRNA (142,201). Three of the four rRNAs (18S, 5.8S, and 25S) are transcribed as a single large pre-rRNA by RNA polymerase I (RNA pol I), whereas the fourth rRNA (5S) is independently transcribed as a pre-rRNA by RNA pol III (201). All four rRNAs are encoded by a 9.1-kb rDNA unit, which is repeated 100 to 200 times on the long arm of chromosome XII (Fig. 1A). In the 35S pre-rRNA, which is the longest detectable precursor, the mature rRNA sequences are separated by two internal transcribed spacer (ITS) sequences, ITS1 and ITS2, and flanked by two external transcribed spacer (ETS) sequences, a 5Ј ETS and a 3Ј ETS (Fig. 1). The 35S pre-rRNA differs from the primary RNA pol I transcript at its 3Ј end because transcription termination maps to nucleotide position ϩ210 relative to the 3Ј end of the mature 25S rRNA, while the 35S pre-rRNA is extended by 7 to 10 nucleotides (78,187,189). Maturation of the 35S pre-rRNA, which contains 10 known processing sites, is a multistep pathway that requires many different trans-acting factors (Fig. 1B and its legend) (97,174,193). Processing of the pre-5S rRNA is independent of 35S pre-rRNA maturation and kinetically faster than formation of mature 18S, 5.8S, and 25S rRNAs (146). The 5Ј end of the mature 5S rRNA corresponds to that of the primary transcript, whereas the 3Ј end is processed from a pre-5S rRNA that is extended by 7 to 13 nucleotides (141). Many specific nucleotides within the rRNA also undergo, mainly shortly after transcription, covalent modification. These modifications include isomerization of uridine to pseudouridine (⌿) by base rotation (45 modified nucleotides), methylation of the 2Ј-hydroxyl group of sugar residues (2Ј-O-ribose methylation; 55 modified nucleotides), and base methylation (about 10 modified nucleotides) (9,21,84,120,135...

The dynein-related AAA ATPase Rea1 is a preribosomal factor that triggers an unknown maturation step in 60S subunit biogenesis. Using electron microscopy, we show that Rea1's motor domain is docked to the pre-60S particle and its tail-like structure, harboring a metal ion-dependent adhesion site (MIDAS), protrudes from the preribosome. Typically, integrins utilize a MIDAS to bind extracellular ligands, an interaction that is strengthened under applied tensile force. Likewise, the Rea1 MIDAS binds the preribosomal factor Rsa4, which is located on the pre-60S subunit at a site that is contacted by the flexible Rea1 tail. The MIDAS-Rsa4 interaction is essential for ATP-dependent dissociation of a group of non-ribosomal factors from the pre-60S particle. Thus, Rea1 aligns with its interacting partners on the preribosome to effect a necessary step on the path to the export-competent 60S subunit.