; the large 60S subunit is composed of three rRNAs and 49 ribosomal proteins (RLS). The assembly of translation competent 80S ribosome is a complex journey that starts in the nucleolus/nucleus and ends in the cytoplasm, and along the way, involving more than 200 non-ribosomal, trans-acting factors. Ribosome biogenesis is an essential and one of the most energy-consuming cellular events. The yeast has been the model system for the studies of ribosome biogenesis but many aspects are still not well defined. How it takes place in humans is only beginning to be unraveled, showing core similarities but also differences. Recent work identifies a very large panel of human genes required for ribosome processing, a subset of which do not have an yeast ortholog. The ribosome biogenesis in higher eukaryotes, as currently known, is described here.
The initial stage of ribosome biogenesis is the transcription of ribosomal RNA (rRNA) genes. RNA polymerase I transcribes a large rRNA precursor that contains the mature 18S, 5.8S and 28S rRNAs, separated by internal (ITS) and flanked by external transcribed spacers (ETS). Of the three, 18S is the rRNA of the 40S subunit; the 5S rRNA component of the 60S subunit is transcribed by RNA polymerase III. As is transcribed, pre-rRNA associates co-transcriptionally with a subset of ribosomal and trans-acting proteins, with small nucleolar ribonucleotide particles (snoRNPs), folds and is subject to modifications while several endo- and exo-nucleolytic cleavage reactions will sequentially eliminate the spacers. Pre-rRNA maturation - the removal of internal and external spacers, is embedded within ribosome biogenesis. It starts with processing at the 5'-ETS site(s); the cleavage of an initial 90S pre-ribosome in the ITS1 spacer, produces the pre-40S and pre-60S ribosomal particles. From this point on, the two pre-ribosomal particles follow independent biogenesis and transport routes. The pre-40S undergoes relatively few compositional changes and is rapidly transported to the cytoplasm. In contrast, pre-60S associates with many trans-acting factors which, at various points during its biogenesis, are released and recycled. Site-specific pseudouridine formation and 2'-O-methylation of residues in regions important for ribosome function, are carried out by small nucleolar RNPs (snoRNPs). The presence of some is also required for initial cleavage reactions. The pol III transcribed 5S pre-rRNA is subject to 3'-exonuclease activity and assembles with a number of binding partners before joining the pre- 60S subunit either immediately delivered to the ribosome or arriving from a cytoplasmic pool. The ribosomal proteins of the small and large subunits assemble in a hierarchical fashion. Remodeling events along the ribosome maturation routes rely on the action of ATPases, GTPases, helicases, kinases and other protein classes. The two ribosomal subunits, independently exported to the cytoplasm, undergo final maturation. The transcriptionally competent 80S ribosome requires 40S, heavily regulated during the initiation phase of translation and 60S, recruited late during initiation, once the mRNA start codon has been located. Along the way, surveillance mechanisms assure its proper assembly, and the structural and functional integrity or, if necessary, its degradation. Defects in components of ribosome biogenesis result in a number of diseases collectively known as ribosomopathies. Several steps in the pathway are described.

rRNA cleavage and processing
The large pre-rRNA: 5' - ETS - 18S rRNA - ITS1 - 5.8S rRNA - ITS2 - 28S rRNA - 3'-ETS (see top of diagram). Both 5'-ETS and ITS contain several cleavage sites within and alternative routes may be followed (details not shown in diagram). Cleavage and processing of 5¿-ETS and at ITS1 sites mark the early stages of 18S rRNA formation. Factors involved in 5'-ETS processing include small nucleolar RNA particles (snoRNPs), ribosomal proteins and non-ribosomal proteins, exonuclease activities. Many have equivalents to components of the small subunit (SSU) processome identified in yeast.
ITS1 processing is dependent upon several factors of which some are also involved in 5'-ETS processing while others are specific. An important aspect of this step is the separation of 90S into the pre-ribosomal 40S and 60S subunits.
In the maturation of pre-60S, endo-nucleolytic processing of ITS2 and 3'-5' and 5'-3' exonuclease activities give rise to 5.8S and 28S (25S in yeast) rRNAs. Factors involved also include components of the exosome (catalytic Dis3 and associated factors Mpp6, C1D and Sklv2l2 known as Mtr4); some factors also play a role in earlier cleavage/processing steps.
Final cleavage reactions occur in the cytoplasm, carried out by Nob1 and Eri1 for the 40S and 60S particles, respectively.

Modification reactions
Site-specific pseudouridine formation and 2'-O-methylation of residues in regions are important for ribosome function; they are carried out by box H/ACA and box C/D small nucleolar ribonucleoproteins (snoRNPs), respectively. The snoRNAs base-pair with target rRNAs and 'guide' the proteins which organize and position the RNA and assure its proper modification.
U17 is the box H/ACA snoRNA; it contains a pseudouridylation loop within a hairpin structure to an H or ACA box. Four core proteins are within the complex: Nhp2, Nop10, Gar1 and dyskerin in humans, of which dyskerin is the pseudouridylase that catalyzes the uridine to pseudouridine (5-ribosyluracil) conversion. First, the nitrogen-carbon bond that links the base to the sugar (N1-C1') is broken; rotation of the base is followed by formation of a new carbon-carbon bond between the base and the sugar (C5-C1'). An extra hydrogen-bond donor distinguishes pseudouridine from other nucleotide. In eukaryotes, box H/ACA is a double hairpin with an H box in the hinge region and ACA box in the tail region, each with a set of core proteins.
Box C/D snoRNAs contain a C/D motif at their termini and an internal C'/D' motif and each assembles with two copies of the core proteins. Nop56 and Nop58, Nhpx and fibrillarin are the core proteins of which fibrillarin is the 2'-O-methyltransferase. U3 and U14, and U8 and U13 are C/D snoRNAs.
Of note is that both types of complexes also play a role in the cleavage/processing of rRNA.
SnoRNAs are transcribed by RNA polymerase II from introns or independently and the biogenesis of snoRNPs, from processing to assembly and localization, involves the concerted action of many factors.

Ribosomal proteins
Ribosomal proteins are specific for the small and the large subunits and they fulfill important roles in the structural assembly and pre-rRNA processing, nuclear export and proper folding of ribosomal particles. Their assembly along the route of ribosome maturation is hierarchical. While most findings come from yeast studies, experiments carried using mammalian systems including human, point to a similar hierarchical assembly. For instance, for the human ribosomal proteins of the small subunit (RPS,40S), using silencing RNA (siRNA) to target them, two functional groups were identified. One group includes RPSs that assemble with pre-RNA, likely co-transcriptionally, in the early steps of 40S maturation - the 'initiation RPSs' and the other group, required for the later steps, including transport and cytoplasmic processing, includes the 'progression RPSs' (some intermediate stages within). In yeast, the ribosomal proteins of the large subunit (RPL, 60S), show a hierarchical assembly, with various members specifically associated with the early, middle and late stages of 60S maturation. The complement of human RPS and of RPL proteins is evidenced in the structures of 80S ribosome solved by cryo electron microscopy.

Other trans-acting factors
In addition to the box C/D and H/ACA modifying enzymes described above, other factors such as ATPases, GTPases, and helicases, among others, participate in ribosome maturation and biogenesis (most involve 60S biogenesis). Their association and release, activity and role have best been characterized in yeast. It is believed that dissociation of non-ribosomal trans-acting factors is mediated by the activity of these power-consuming entities. For instance, the remodeling activity of Mdn1 ATPase (known as Rea1) within pre-60S may affect the activity of Gnl2 GTPase, among others. Gnl2, whose site overlaps with the export adapter Nmd3, possibly prevents the transport of an 'immature' 60S subunit. Mdn1 mediated structural rearrangements promote the K+-dependent GTPase activity of Gnl2 leading to its release from pre-60S and clearing the way to 60S export.
RNA helicases bind and remodel RNA and RNPs; the ATP-dependent helicase activity unwinds double-stranded RNA and also as act as RNA clamps (annealing). The role of DExD/H-box RNA helicases in both 40S and 60S biogenesis has been evidenced in yeast; the role of mammalian enzymes has been documented for some. Examples include Ddx10, Ddx18, Dhx15 and Dhx37 in 40S biogenesis, Ddx24 and Ddx56 in 60S biogenesis.
Several kinases have been shown to be involved in the maturation of 40S subunit and the recycling of its trans-acting factors. Some trans-acting factors are involved in the maturation of both particles. Screening of a large panel of human nucleolar proteins identifies a substantial set of processing factors important for ribosome biogenesis. While a good fraction of genes have an yeast ortholog and many exhibit a similar function, a subset of genes have additional or alternative functions, others do not have an ortholog in yeast [check PMID: 23973377, Figure2 and supplemental information].

Nuclear export/transport to the cytoplasm
The transport of pre-40S and pre-60S occurs independently of each other but for both subunits, it is dependent on the GTP/GDP cycle of Ran. Most of what is known comes from yeast studies with relatively little information for the transport of the two subunits in higher eukaryotes. The nuclear export factor Xpo1 (known as Crm1) is required for both pre-40S and pre-60S subunits; pre-60S can also be exported by Xpo5. The adapters for Xpo1 mediated transport appear to be specific for either subunit. Nmd3 is the only adapter for pre-60S subunit while the adapters for pre-40S, better known in yeast, include Ltv1, also involved in pre-40S maturation. Several nucleoporins such as Nup214 and Nup88 are important for transport of human pre-60S; the FG-repeat region of Nup214 acting as binding site for Xpo1. Additional factors found only in higher eukaryotes could play a role in the transport of ribosomal particles based on function and/or location; their involvement remains to be established.

Cytoplasmic maturation, 80S formation, links to translation initiation
Upon nuclear export, the 18S rRNA in 40S and the 5.8 rRNA in 60S undergo final maturation steps carried out by Nob1 and Eri1, respectively. In addition, non-ribosomal proteins that have travelled to the cytoplasm and export factors must be released/recycled.
The formation of 80S is intimately connected to the initiation of translation. The 40S, in association with other factors and part of a 48S initiation complex, scans for the initiating codon (ATG), leading to recruitment of 60S and formation of 80S. Eif6 binds 60S at the interface between 40S and 60S preventing their association. The two subunits can interact in the absence of mRNA forming an inactive 80S ribosome. Phosphorylation of Eif6 leads to its dissociation and formation of the 80S which will then translate and elongate the nascent polypeptide. Gnb2l1, known as Rack1 and an adaptor for PKC enzymes in PKC signaling, binds the 40S ribosomes. Interaction with Eif6 and recruitment of PKC would then promote Eif6 phosphorylation and dissociation from 60S.

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