Eukaryotic Ribosome Assembly and Export

Eukaryotic Ribosome Assembly and Export VG Panse and CS Weirich, ETH Zürich, Switzerland r 2016 Elsevier Inc. All rights reserved.

Introduction The ribosome is responsible for carrying out the final decoding step in gene expression, in which mRNA is translated into proteins. The overall structure and function of this molecular machine is universally conserved and consists of two subunits composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). The small subunit, which is 40S in eukaryotes and 30S in bacteria and archae, is responsible for codon recognition and pairing mRNAs with their cognate tRNAs (Wimberly et al., 2000). The large subunit, which is 60S in eukaryotes and 50S in bacteria and archae, catalyzes the peptidyl transferase reaction to synthesize polypeptide chains (Ban et al., 2000). Biogenesis of these enormous ribonucleoprotein complexes is a critical and energy-consuming task for all cells. Although the overall architecture of the ribosome is conserved, eukaryotic ribosomes are significantly larger than their prokaryotic counterparts and contain proportionally more rRNA (65%) and less r-protein (35%) mass. The large, 60S subunit of Saccharomyces cerevisiae contains three rRNAs (25S, 5.8S, and 5S) and 46 r-proteins. The small, 40S subunit contains a single rRNA (18S) and 33 r-proteins (Ben-Shem et al., 2011; Klinge et al., 2011; Rabl et al., 2011). The structures of both ribosomal subunits have been solved, providing an exquisite level of detail regarding their architectures and unprecedented insights into the mechanism of translation (Ramakrishnan, 2014). Despite these advances, we still understand relatively little about the assembly and transport pathways required to generate these molecular machines. This article will focus on what is currently known and will focus on budding yeast, which has been used to generate a general framework to further dissect this conserved biosynthetic pathway.

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Eukaryotic ribosome assembly takes place in multiple cellular compartments: the nucleolus, the nucleoplasm, and the cytoplasm (Figure 1). This assembly process is a complex task that requires the coordination of RNA polymerases I, II, and III, as well as an efficient splicing machinery and efficient intracellular transport machinery. Surprisingly, despite this complexity, ribosome biogenesis is an incredibly efficient process, with yeast cells producing up to 60 ribosomes per second (Warner, 1999; Tschochner and Hurt, 2003).

Ribosomal Pre-rRNA Processing Ribosome biogenesis begins co-transcriptionally on the 35S pre-rRNA (45S in mammals), which is synthesized by RNA Polymerase I (Figure 2; Dragon et al., 2002; Woolford and Baserga, 2013). This transcript contains both 5’ and 3’ externally transcribed spacer (ETS) sequences, as well as the three rRNAs (18S, 5.8S, and 25S), separated by the internally transcribed spacer sequences ITS1 and ITS2. The yeast 90S particle (which is homologous to the mammalian SSU (Small SubUnit processome) assembles on this precursor rRNA, forming structures first described by electron microscopy as ‘Christmas trees’ forming at the rDNA loci of Xenopus oocytes (Miller and Beatty, 1969; Dragon et al., 2002; Grandi et al., 2002). This precursor is cleaved first at site A0, generating the shorter 33S pre-rRNA. The second cleavage event occurs at site A1, generating the 32S pre-rRNA. The third cleavage site occurs at site A2 and generates the 20S and 27SA2. At this point, the 20S prerRNA is released to be further processed within the context of the 43S, which will become the mature 40S subunit, and is

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35S rRNA Figure 1 Eukaryotic ribosomes assemble along pathways that span the nucleolus, nucleoplasm, and cytoplasm. The 90S particle (orange) is built from the 35S pre-rRNA, which recruits assembly factors and small subunit r-proteins co-transcriptionally. Cleavage at site A2 separates the 90S into a pre-40S subunit (green) and a pre-60S subunit (blue), which follow independent maturation pathways. Assembly factors bind and drive maturation steps on pre-ribosomal particles, then are sequentially released. Final maturation of both the 40S and 60S pre-ribosomal subunits occurs in the cytoplasm. Question marks indicate uncertainty in the assembly pathway.

Encyclopedia of Cell Biology, Volume 3

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Figure 2 The pre-rRNA processing pathway of pre-ribosomal particles. pre-rRNA sequences that are part of the 60S subunit (blue, red) and the 40S subunit (green) are indicated. These sequences are processed away by ITS and ETS sequences (black lines) by nucleases (names in red). Both processing pathways of the 27AS2 pre-rRNA are shown. Note that these pathways generate two rRNAs of slightly different lengths, both of which are incorporated into the 60S. Final processing of the 6S pre-rRNA to mature 5.8S rRNA takes place in the cytoplasm. Red question marks indicate that the enzyme mediating the indicated cleavage step has not been identified.

eventually processed in the cytoplasm at site D to generate the mature 18S rRNA (Kressler et al., 1999). The remaining particles, containing 27AS2 pre-rRNA, recruit the 5S rRNA, which is transcribed by RNA polymerase III and bound by the biogenesis factors Rpf2 and Rrs1 and the ribosomal proteins uL18 (human L5) and uL5 (human L11) (Zhang et al., 2007; nomenclature as in Ban et al., 2014). Then, 27AS2 continues processing through one of the two pathways. The first pathway, comprising 85% of molecules, begins with

cleavage at site A3 in ITS1 by the RNase MRP, followed by trimming to site B1S by the exonuclease Rat1. The second pathway, which processes the remaining 15%, begins with cleavage directly at site B1L and is followed by cleavage at B2, forming the end of the 25S concomitantly with cleavage at site B1 (Kressler et al., 1999). In addition to RNA cleavage events, eukaryotic ribosomes undergo multiple posttranslational RNA modifications, including isomerization of uridines to pseudouridines and

Cell Division/Death: Regulation of Cell Growth: Eukaryotic Ribosome Assembly and Export

methylation (Woolford and Baserga, 2013; Lafontaine, 2015). Two classes of small nucleolar RNAs (snoRNAs) direct these modifications: the box C/D snoRNAs direct methylation, whereas the box H/ACA snoRNAs direct pseudouridylation. Although both modifications are more widespread in eukaryotes, occurring between 50 and 100 times each, they are also found in prokaryotes and archae. The functions of individual modifications remain unclear, and have been proposed to affect translational efficiency and rRNA structure (BaxterRoshek et al., 2007).

Trans-Acting Factors Involved in Ribosome Assembly Unsurprisingly, ribosomal proteins themselves play critical roles in ribosome assembly. One study demonstrated that most 40S subunit proteins interact directly with the 35S prerRNA, though the interactions are weak. In addition, the small subunit proteins uS17 (yeast Rps11), uS4 (yeast Rps9), and uS15 (yeast Rps13) are required for early rRNA processing events and are homologous to the Escherichia coli ‘primary binders’ that mark the beginning of prokaryotic ribosome assembly in vitro (Held et al., 1974). However, unlike their prokaryotic counterparts, eukaryotic ribosomes cannot selfassemble in vitro and require, in addition to ribosomal proteins, 4200 additional trans-acting biogenesis factors. Genetic screens in budding yeast and, in particular, the advent of tandem affinity purification (TAP) have yielded a long list of factors involved in ribosome assembly and export (Tschochner and Hurt, 2003). The majority of uncovered factors are essential, contrasting sharply with ribosome biogenesis in prokaryotes, which does not rely on essential biogenesis factors (Hage and Tollervey, 2004; Connolly and Culver, 2009). These evolutionarily conserved factors are generally thought to play roles in distinct maturation stages, after which they are released and recycled. In addition to scaffolds and RNA-interacting proteins, energy-consuming enzymes such as AAA-adenosine triphosphatase (ATPases), ABC-ATPases, guanosine triphosphatase (GTPases), and ATP-dependent RNA helicases are thought to be responsible for factor release, conferring directionality and irreversibility to the assembly process. Recent studies have begun to unravel the substrates, pre-ribosomal binding sites, and specific functions of these energy-consuming enzymes (Kressler et al., 2012; Strunk et al., 2012). Two well-characterized examples of energy-dependent factor release are the essential AAA-ATPases Rix7 and Rea1, both required during 60S biogenesis. A related AAA-ATPase Drg1 is required for cytoplasmic maturation of pre-60S subunits (see Section Cytoplasmic Maturation of the 60S Subunit). Rix7, an early acting energy-consuming release factor, is responsible for early nucleolar 60S maturation. Although clear evidence remains elusive, this ATPase is proposed to be involved in the release of the assembly factor Nsa1. In turn, the release of Nsa1 is thought to require posttranslational modifications, which allow its recognition by Rix7. This energy-consuming release step is critical to facilitate the nucleolar to nucleoplasmic movement of 60S pre-ribosomal particles (Kressler et al., 2008; Panse et al., 2006). Rea1 is involved at multiple maturation steps of the large ribosomal precursor (Ulbrich et al., 2009; Bassler et al., 2010;

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Kressler et al., 2012). Rea1 triggers the release of the biogenesis factors Ytm1 and Rsa4 (Nissan et al., 2004; Ulbrich et al., 2009; Bassler et al., 2010). Release of Ytm1, along with its cofactors Erb1 and Nop7, has been proposed to trigger the displacement of neighboring assembly factors, conferring directionality to the ribosome maturation process (Nissan et al., 2002, 2004; Ulbrich et al., 2009). In addition to its nucleolar role, recent studies suggest that Rea1 is involved in a pre-60S nuclear checkpoint, releasing the GTPase Nug2 (Nog2) and allowing recruitment of the 60S export factor Nmd3. These observations suggest that Rea1 coordinates nuclear maturation and export of pre-60S particles (Matsuo et al., 2014). Many 40S biogenesis factors, such as the RNA dimethylase Dim1p, Dim2p, Enp1p, Hrr25p, Nob1p, the RNA helicase Prp43p, Rrp12p, Tsr1p, and Tsr2, required for 40S biogenesis are present within the 90S prior to A2 cleavage and release of the 20S pre-rRNA (Panse and Johnson, 2010). After A2 cleavage, additional proteins, including Ltv1p, Pfa1p/Sqs1p, Rio1p, and Rio2p, join the 20S pre-rRNA assembled within the pre-40S subunit (Panse and Johnson, 2010). Although the mechanisms by which these factors are recruited and the precise roles they play during biogenesis remain unclear, some details have been identified. For example, the kinase Hrr25 is thought to phosphoryate Enp1p, Ltv1p, and the small subunit protein uS3 (yeast Rps3p), perhaps to cause a structural change in these pre-40S particles and promote efficient transport through the nuclear pore (Schafer et al., 2006). In addition, the essential RNA dimethylase Dim1p is required for early nucleolar processing events, though it also plays a later role in the cytoplasm (Lafontaine et al., 1994, 1998) (see Section Cytoplasmic Maturation Events of the 40S Subunit). In addition to assembly factors and energy-consuming release and remodeling factors, pre-ribosomal particles have been proposed to require the action of r-protein delivery factors, termed ‘escortins.’ One escortin, Tsr2, releases eS26 from its import receptor in a RanGTP-independent manner and then ensures its secure transfer to the earliest identified 90S pre-ribosome particle (Schutz et al., 2014). Given the large influx of r-proteins into the nucleus and the inherent instability of these RNA-binding polypeptides, a network of escortins could keep r-proteins soluble until they are incorporated into pre-ribosomes.

Nuclear Export of Pre-Ribosomal Particles Although translation is a cytoplasmic event, ribosome production begins in the nucleolus. Therefore, in addition to the import and nucleolar targeting of ribosomal proteins, ribosomal subunits must also be exported into the cytoplasm. All nucleocytoplasmic transport takes place through nuclear pore complexes (NPCs), which are macromolecular assemblies that span the double lipid bilayer of the nuclear envelope. The extraordinary size of pre-ribosomal particles (approximately 2 MDa) presents a particular challenge to the transport machinery (Ribbeck and Gorlich, 2002). In addition, the highly charged surfaces of these complexes must be shielded from the hydrophobic FG-repeats present within the NPC. Despite these obstacles, ribosome export is remarkably efficient, with growing yeast cells exporting about 25 pre-ribosomal particles

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per minute per NPC (Warner, 1999). How the transport machinery manages to move these large, charged pre-ribosomal particles quickly and efficiently has been a topic of intense study, yielding the identification of multiple export factors, as described below. In the 1990s, visualization assays based on green fluorescent protein (GFP)-tagging of ribosomal proteins and in situ hybridization against rRNAs were used to identify factors involved in pre-ribosomal subunit export (Hurt et al., 1999; Moy and Silver, 1999; Stage-Zimmermann et al., 2000; Milkereit et al., 2003). These approaches identified several structural components of the NPC, the small GTPase Ran, Ran regulators, and the export receptor Crm1/Xpo1 as export factors for pre-60S and pre-40S subunits (Hurt et al., 1999; Moy and Silver, 1999; Stage-Zimmermann et al., 2000). However, these factors are not predicted to be sufficient for pre-ribosomal subunit export, since the rate of transport receptor shuttling is known to decrease as the size of a cargo increases (Ribbeck et al., 1998). Transport of very large pre-ribosomal particles is predicted to be extremely slow, suggesting the requirement of multiple export factors to increase translocation rates. Visual and genetic screens identified the nuclear export signal (NES)-containing adaptor Nmd3, which forms a trimeric complex with RanGTP and Xpo1 on the surface of the pre-60S subunit (Ho et al., 2000; Gadal et al., 2001). Using parallel approaches based on systematic RNA interference (RNAi) and ribosomal protein reporters, such visual screens have been extended to human cells. One such screen uncovered an essential role of the RanGTP-binding protein Exp5 as an export receptor for pre-60S subunits (Wild et al., 2010). A structural modeling approach identified an additional export factor Rrp12, which binds both pre-ribosomal subunits and FXFG-repeat nucleoporins (Oeffinger et al., 2004). Like RanGTP-dependent export factors, Rrp12 contains HEAT repeats, and therefore may interact with FXFG repeats using a similar mechanism. Intriguingly, Rrp12 was shown to interact with both GTP and guanosine diphosphate (GDP) forms of Ran. However, the mechanism by which Rrp12 promotes ribosomal subunit export and its regulation by Ran remain unknown. Intriguingly, the essential mRNA export factor Mex67-Mtr2 (TAP-p15 in humans) also exports both pre-60S and pre-40S subunits (Segref et al., 1997; Yao et al., 2007; Faza et al., 2012). The large subunit of the heterodimer, Mex67, consists of an Nterminal domain, a leucine-rich repeat (LRR), a nuclear transport factor 2 (NTF2)-like middle domain, and a C-terminal ubiquitin-associated (UBA-like) domain (Strasser et al., 2000). Mtr2 shares structural features with NTF2 (Bayliss et al., 2002) and heterodimerizes with the NTF2-like middle domain of Mex67 (Santos-Rosa et al., 1998; Strasser et al., 2000). Although Mex67 and Mtr2 do not rely on the RanGTP system, and have no structural similarities to karyopherins, the heterodimer also exports cargoes by interacting with FG-rich nucleoporins. To export mRNAs and pre-ribosomal subunits, Mex67-Mtr2 uses different binding surfaces. Interaction with pre-ribosomal subunits relies on loops within the NTF2-like domains of Mex6 and Mtr2 (Fribourg and Conti, 2003; Senay et al., 2003; Yao et al., 2007, 2008). Whether these loops directly interact with an exposed structured rRNA or a protein factor(s) on pre-ribosomal subunits still remains to be

determined. The NTF2-like domains of Mex67-Mtr2 and the UBA-like domain of Mex67 interact with FG-repeat nucleoporins to mediate mRNA and pre-ribosomal subunit export (Strasser et al., 2000; Strawn et al., 2001). The mRNA export factor Npl3 also plays a role in pre-60S subunit nuclear export (Hackmann et al., 2011). Thus, the mRNA export machinery is partially adapted to carry both pre-ribosomal and mRNA cargoes. It is not yet known how the cellular pools of these factors are fractionated to participate in the nuclear export of mRNAs and pre-ribosomal subunits. Understanding the molecular basis of this allocation will reveal how the three export pathways cross-talk to deliver appropriate levels of mRNA and ribosomal subunits in the cytoplasm. In addition to essential transport receptors, pre-ribosomal particles are also associated with nonessential transport factors that directly facilitate their translocation through the NPC channel, through interactions with nucleoporins. Although these factors are known to promote pre-ribosomal subunit export, it is not known whether they decorate every subunit during its export process, adding incrementally to export efficiency, or whether each subunit requires a quorum of factors, but not all, for efficient export. Two examples of these nonessential factors are described below. The pre-60S export factor Arx1 is structurally homologous to methionine amino peptidases, which remove N-terminal methionines from nascent polypeptides as they emerge from the ribosome (Bradatsch et al., 2007; Hung et al., 2008). However, Arx1 lacks methionine amino peptidase activity, and mutations in the methionine-binding pocket impair pre-60S subunit export (Bradatsch et al., 2007). When bound to the pre-60S, Arx1 occludes the exit tunnel of the 60S subunit and stabilizes the eukaryotic-specific rRNA expansion segment 27 (ES27) in an alternate conformation (Bradatsch et al., 2012; Greber et al., 2012). However, the details of how Arx1 mediates pre-60S export, as well as the function of its interaction with ES27 currently remain unknown. The mRNA export factor Gle2 also mediates the export of pre-60S subunits, using a distinct, non-FG repeat binding mechanism (Occhipinti et al., 2013). This factor interacts with late pre-60S subunits as well as the Gle2-binding-sequence (GLEBS)-motif of the nucleoporin Nup116. The Gle2-Nup116 interaction is required both to bind and export pre-60S subunits, but disruption of this interaction has no effect on mRNA export. Therefore, Gle2 appears to have dual functions in mRNA export and pre-60S export. This unique mechanism has been proposed to prevent kinetic delays during pre-60S translocation through the NPC, especially if cargos have failed to recruit their optimal complement of export factors. In addition to Gle2 and Arx1, Ecm1 and Bud20 have also been identified as factors involved in pre-60S biogenesis (Bradatsch et al., 2007; Yao et al., 2010; Altvater et al., 2012; Bassler et al., 2012). One major challenge in the field remains to identify the binding sites of individual export factors on preribosomal particles. Together, these analyses will provide insight both into the structure and of pre-ribosomal export particles and the diversity underlying this export process. Export of pre-40S subunits also relies on auxillary factors such as the Xpo1 interacting proteins Ltv1, Dim2, and Rio2 (Zemp et al., 2009). In addition, the conserved RanGTPbinding protein Yrb2 (RanBP3 in humans) has been identified

Cell Division/Death: Regulation of Cell Growth: Eukaryotic Ribosome Assembly and Export

as a specific pre-40S export factor (Taura et al., 1998; Moy and Silver, 2002). The identification of multiple, nonessential NES containing adaptors suggests that these factors play redundant roles to recruit the essential export receptor Xpo1. Such redundancy would act to guarantee Xpo1 recruitment, but remains to be demonstrated.

Cytoplasmic Events in Ribosome Biogenesis Although the majority of ribosome biogenesis takes place in the nucleus, both subunits complete their maturation only in the cytoplasm. Completing ribosome biogenesis in this subcellular compartment potentially allows a system of checkpoints to ensure that only functional ribosomal subunits are allowed to participate in translation.

Cytoplasmic Maturation of the 60S Subunit The cytoplasmic maturation pathway of the 60S subunit requires the release of multiple factors in a specific order (Figure 3; Lo et al., 2010). These release events rely on factors present exclusively in the cytoplasm that associate only transiently with pre-60S particles (Panse and Johnson, 2010). The cytoplasmic maturation pathway begins with the release of Rlp24 by Drg1. Next, the ribosomal protein eL24 (yeast Rpl24) is incorporated onto the subunit and recruits Rei1. Together with Jjj1 and the ATPase Ssa1/Ssa2, these proteins cause subsequent Arx1 and Alb1 release. Only after the release of these two proteins, as well as formation of the acidic ribosomal stalk and correct assembly of the catalytic P-site, is the maturation factor Tif6 also released (Bussiere et al., 2012). The AAA-ATPase Drg1 initiates the earliest cytoplasmic maturation step on pre-60S particles, which is required for subsequent steps that release and recycle additional factors. Consequently, Drg1 catalytic mutants accumulate shuttling assembly factors Rlp24, Nog1, Arx1, and Tif6 on cytoplasmic pre-60S subunits (Pertschy et al., 2007). A recent study demonstrated that Drg1 catalyzes Rlp24 dissociation, which is

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essential for subsequent release events (Kappel et al., 2012). Drg1 is recruited to a C-terminal region within Rlp24 that also stimulates its ATPase activity. Notably, Rlp24 release also appears to require the FG-nucleoporin Nup116. Thus, Drg1 has been hypothesized to provide irreversibility to the nuclear export step and initiate the cytoplasmic maturation pathway. Release of the ribosomal-like protein Rlp24 is necessary to allow the ribosomal protein eL24 to assemble into the pre-60S subunit. This exchange allows recruitment of the zinc-finger protein Rei1, which triggers the next maturation event. Rei1 works in conjunction with the J-domain protein Jjj1 and the ATPase Ssa1/Ssa2 (Hsp70) to release Arx1 and its interacting partner Alb1 (Demoinet et al., 2007; Meyer et al., 2007). Interestingly, these data indicate that Arx1 has an inhibitory role in driving cytoplasmic maturation pathway of pre-60S subunits. Arx1 has evolved from methionyl amino peptidases (MetAPs) (Bradshaw et al., 1998). Based on similarity of Arx1 to MetAPs, Arx1 was predicted to bind to the MetAP site on the ribosome, preventing MetAP binding. Indeed, Cryo-EM studies showed that Arx1 binds near the ribosomal protein uL25, which interacts with the signal recognition particle and the translocon, further suggesting that Arx1 plays an inhibitory role in the cytoplasmic maturation pathway (Dalley et al., 2008). Tif6 is another biogenesis factor that must be removed from the large subunit in the cytoplasm. This shuttling factor remains bound to cytoplasmic pre-60S particles, preventing their interaction with mature 40S subunits (Russell and Spremulli, 1979; Valenzuela et al., 1982). The GTPase Efl1 and its cofactor, the Shwachman–Bodian Syndrome protein Sdo1, trigger release of TIf6 (Becam et al., 2001; Senger et al., 2001; Menne et al., 2007). In efl1 and sdo1 mutants, Tif6 accumulates in the cytoplasm on late pre-60S subunits, and tif6 mutations that weaken its affinity for the 60S subunit suppress the growth defects of efl1 and sdo1 mutants, providing strong genetic evidence that Tif6 is the primary substrate of Efl1 and Sdo1. Interestingly, Tif6 also mislocalizes in yvh1 mutants, which also display defects in stalk assembly (Kemmler et al., 2009;

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Figure 3 Cytoplasmic events required for 60S pre-ribosome maturation. Export factors are shown in yellow, and shuttling assembly factors are shown in green. Release of assembly factors Nog1 and Rlp24 by the ATPase Drg1 is thought to be the first step in the 60S pre-ribosome cytoplasmic maturation pathway. Rei1, J-domain protein Jjj1, and the ATPase Ssa1/Ssa2 are required for the release of Arx1 and Alb1. Stalk assembly relies on release of Mrt4 by Yvh1 and recruitment of uL10, which releases Yvh1. Tif6 release by Efl1 and Sdo1, as well as Nmd3 release by Kre35 follow stalk assembly. Factors that are released by unknown mechanisms (?) are listed in gray.

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Lo et al., 2009). During translation, the stalk functions in recruitment and activation of the GTPase eEF2 (Bargis-Surgey et al., 1999). Given that Efl1 is closely related to eEF2, stalk assembly might play a similar role in biogenesis, recruiting Efl1 for the release of Tif6. These analyses suggest that the cytoplasmic maturation events in the 60S biogenesis are coupled and ordered sequentially from Drg1-dependent release of Rlp24 to Efl1-dependent release of Tif6 (Figure 3). Therefore, this pathway appears to rely on the coupled stepwise recruitment of cytoplasmic release factors to pre-60S subunits. The essential NES containing adapter Nmd3 must also be released from pre-60S subunits in the cytoplasm. Both the ribosomal protein uL16 (yeast Rpl10) and the GTPase Kre35 (Lsg1) have been implicated in the release of Nmd3 (Karl et al., 1999; Hedges et al., 2005). Mutations in uL16 prevent release of Nmd3 from pre-60S subunits. Moreover, mutations in Kre35 that are predicted to disrupt its GTPase activity also block Nmd3 release in the cytoplasm. These results suggest that Kre35 triggers the binding of uL16 to the 60S, an event that is coupled to the release of Nmd3. However, the exact timing of Nmd3 release has not been elucidated. Assembly of the stalk structure on the 60S subunit represents a critical step toward ribosome functionality. This structure is essential for the recruitment and activation of translation factors, in particular elongation factors. In yeast, the stalk region is composed of the ribosomal protein uL10 (yeast Rpp0) and two heterodimers of P1 (yeast Rpp1) and P2 (yeast Rpp2) (Berk and Cate, 2007). However, these proteins are not present in pre-60S subunits (Lo et al., 2009; Kemmler et al., 2009). Instead, the ribosomal-like protein Mrt4 is assembled into the pre-60S in the nucleus. Once the pre-60S is exported, the phosphatase Yvh1 catalyzes Mrt4 release, allowing the recruitment of uL10. However, the precise mechanism of Mrt4 release and the molecular events that lead to stalk assembly remains elusive (Lo et al., 2009; Kemmler et al., 2009).

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In addition to these factors, a study combining genetic trapping, affinity purification and a targeted proteomic approach was used to characterize the proteome of 60S preribosomes after nuclear export (Altvater et al., 2012). This hybrid approach identified the factors Bud20, Nug1, Nsa2, and Rli1 as 60S-associated factors that are released in the cytoplasm after Drg1-mediated release of Rlp24. The functional significance of shuttling behavior of the identified assembly factors remains unknown. It could be that they participate directly in the transport and/or final functional proofreading of pre-60S subunits. The factors and mechanisms that release these shuttling assembly factors from 60S particles in the cytoplasm remain to be discovered.

Cytoplasmic Maturation Events of the 40S Subunit The small pre-ribosomal subunit is accompanied to the cytoplasm by a handful of proteins, including Enp1, Tsr1, Ltv1, Dim1, Dim2, Nob1, Rio2, Hrr25, and Prp43 (Figure 4). These factors mediate both export and endo-nucleolytic cleavage of 20S pre-rRNA to 18S rRNA. This final cleavage event is an essential cytoplasmic maturation step that renders pre-40S particles translation-competent (Strunk et al., 2011). How these shuttling factors are released from pre-40S subunits in the cytoplasm remains unclear. Prior to 20S pre-rRNA cleavage, two adenine bases near the 3’-end of the future 18S rRNA are dimethylated by the essential dimethylase Dim1 (Lafontaine et al., 1994). However, this modification is not essential, since processing of 20S pre-rRNA to 18S rRNA can still occur in the absence of dimethylation, forming functional 40S subunits. Dimethylation was suggested to play a role in fine-tuning of translation, as the catalytic inactive dim1 mutant lacking 40S dimethylation displays increased antibiotic sensitivity (Lafontaine et al., 1998). Multiple

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Figure 4 Cytoplasmic events required for 40S pre-ribosome maturation. Export factors are shown in yellow, and shuttling assembly factors are in green. Release of shuttling factors and final pre-rRNA processing requires the ATPase Rio1 and the GTPase eIF5b as well as interactions with a mature 60S subunit. 20S pre-rRNA is processed in the cytoplasm to the final 18S rRNA by the endonuclease Nob1 within a 80S-like particle. Factors that are released by unknown mechanisms (?) are listed in gray.

Cell Division/Death: Regulation of Cell Growth: Eukaryotic Ribosome Assembly and Export

energy-consuming ATPases (Prp43, Rio2, and Fap7) and the PIN-domain endonuclease Nob1 have been implicated in this late maturation (Clissold and Ponting, 2000; Geerlings et al., 2003; Vanrobays et al., 2003; Jakovljevic et al., 2004; Lamanna and Karbstein, 2009; Pertschy et al., 2009). Intriguingly, Nob1 becomes associated with 40S pre-ribosomes in the nucleus, suggesting that this protein is specifically activated in the cytoplasm. This activation of Nob1 in the cytoplasm occurs within an 80S-like particle formed between by the joining of a pre-40S subunit with a mature 60S subunit. Nob1 activation seems to also require the GTPase activity of the translation initiation factor Fun12/eIF5b and the ATPase Rio1 (Lebaron et al., 2012; Strunk et al., 2012; Turowski et al., 2014).

Quality Control Mechanisms for Ribosome Assembly Because accurate protein translation is critical to the cell, preribosomes must undergo quality control prior to their release into the translating pool. Quality control begins in the nucleus, where aberrant pre-ribosomes are recognized and rapidly degraded by the exosome (Mitchell et al., 1997; Allmang et al., 2000). Although recognition depends in part on the poly-adenylating activity of the TRAMP complex, the precise recognition mechanism remains unclear (Dez et al., 2006). A second quality control step is the cytoplasmic nonfunctional RNA decay (NRD) pathway, which targets improperly assembled pre-ribosomal subunits for degradation (LaRiviere et al., 2006; Cole et al., 2009). In addition to these quality control mechanisms, a cytoplasmic pathway exists to proofread pre-ribosomes in the cytoplasm, preventing the premature binding of translation factors and, most likely, actively checking pre-ribosomal subunits for functionality. Cytoplasmic 60S proofreading depends a coterie of factors that block the binding of ribosomal proteins, interactions with translation factors, or pairing with the 40S (Figure 3). Proteins that block r-protein binding include the ribosomal-like proteins Rlp24 and Mrt4, that act as ‘placeholders’ for the r-proteins eL24 and P0, respectively. Tif6 prevents binding of pre-60S particles to mature 40S subunits, ensuring that only properly assembled mature 60S subunits are able to engage their partners. In addition, the GTPase Efl1, which is required for Tif6 release, shares sequence similarity with the GTPase elongation factor eEF2, and has been proposed to check the integrity of the P-site for functionality (Bussiere et al., 2012). Therefore, cytoplasmic release factors appear to be responsible both for releasing shuttling assembly factors and, simultaneously, testing ribosome function. Similar to the 60S, quality control of the 40S relies on assembly factors that are thought to prevent the premature binding of initiation factors, mRNA, tRNA, and the 60S subunit (Figure 4). Based on cryo-EM studies of a late cytoplasmic pre40S particle, Ltv1, Enp1, Rio2, Tsr1, Dim1, Pno1, and Nob1 binding sites have been identified (Strunk et al., 2011). Ltv1 and Enp1 directly bind uS3 (yeast Rps3) on its solvent side, thereby blocking the mRNA channel opening. Rio2, Tsr1, and Dim1 bind the subunit interface, thus preventing joining of the mature 60S subunit and translation initiation factor eIF1A. Nob1 and Pno1 block the binding of eIF3 and thereby interfere with translation initiation. Therefore, these factors are likely to function, in part, by preventing premature translation initiation.

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After the release of Rio2, Tsr1, and Dim1, the resulting pre40S particle has been shown to interact with mature 60S subunits, forming an 80S-like particle in vitro and in vivo (Strunk et al., 2012; Lebaron et al., 2012). This translation-like interaction, which could test the ability of a pre-40S subunit to engage with 60S subunits, triggers the activity of Nob1 to cleave 20S pre-RNA to mature 18S rRNA in vitro. In addition, the conserved translation initiation factor eIF5b/Fun12 is important for formation of the 80S-like particle (Lebaron et al., 2012). After cleavage, the ABC-ATPase Rli1 is thought to cause dissociation of the 80S-like particle and Nob1 release (Strunk et al., 2012). Thus, processing of the 20S pre-rRNA has been proposed to act both as a trigger for subunit maturation and as a quality control mechanism sensing translational competence. By constantly engaging with each other in the cytoplasm, ribosomal subunits may sense their ‘decoding’ ability to segregate and target faulty subunits for disassembly and degradation (Schutz and Panse, 2012).

Concluding Remarks The biogenesis and nuclear export of ribosomal subunits is one of the most important energy-consuming tasks in a eukaryotic cell. The pathways responsible for 60S and 40S biogenesis are remarkably complex, requiring hundreds of evolutionarily conserved assembly factors in addition to the RNA and protein components of the ribosomal subunits themselves. Surprisingly for such a complex pathway, ribosome biogenesis and transport is also incredibly fast and at the same time efficient. Although the factors involved in this process have been largely delineated, their functions remain unclear, and there remains much to be learned about the detailed mechanism by which eukaryotic ribosomes are generated and transported through the cell. We anticipate a combination of genetic, cell-biological, biochemical, and highresolution structural approaches in budding yeast will shed light on this poorly understood fundamental process.

Acknowledgments V. G. Panse is supported by grants from the Swiss National Science Foundation and the ETH Zurich, and is the recipient of a Starting Grant Award (EURIBIO260676) from the European Research Council.

See also: Cell Division/Death: Regulation of Cell Growth: Ribosomal RNA Processing

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Further Reading Fernández-Pevida, A., Kressler, D., de la Cruz, J., 2014. Processing of preribosomal RNA in Saccharomyces cerevisiae. Wiley Interdiscip Reviews, RNA 6, 191–209.

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Relevant Websites http://www.mol.biol.ethz.ch/groups/ban_group/nomenclature ETH Zürich. http://www.yeastgenome.org Saccharomyces Genome Database. http://www.arb-silva.de Silva ribosomal RNA database. http://people.biochem.umass.edu/fournierlab/snornadb/main.php snoRNA database.