In the majority of eukaryotes, one ribosomal protein (RP) of each subunit is encoded as a linear fusion with an N-terminal ubiquitin (Ub). In addition to genome-encoded polyubiquitin chains, these Ub-RP fusion proteins serve as source of cellular ubiquitin, which is liberated from these precursors by endoproteolytic processing (Martín-Villanueva et al., 2021). The released ubiquitin constitutes a major fraction of the ubiquitin pool in both yeast and mammalian cells (Bianchi et al., 2015; Finley et al., 1987; Ozkaynak et al., 1987). From the perspective of the ubiquitin proteasome system, it is not entirely clear why ubiquitin is encoded as a fusion with RPs; but such a configuration seems suited to jointly determine the capacities of the protein degradation and protein synthesis machineries. From the perspective of the fused RPs, their N-terminal ubiquitin moieties seem beneficial as they have been suggested to serve as ‘in cis’ chaperones for folding and solubility of the respective RP partners, as shown by studies in yeast (Finley et al., 1989; Lacombe et al., 2009).

In humans, the ribosomal proteins eS31 (RPS27A) and eL40 (RPL40) of the small and large ribosomal subunit, respectively, are synthesized as Ub-RP precursor proteins (Baker and Board, 1991; Kirschner and Stratakis, 2000; Lund et al., 1985), similar to their yeast homologs (Fernández-Pevida et al., 2016; Finley et al., 1989; Lacombe et al., 2009; Martín-Villanueva et al., 2020). How cleavage of their ubiquitin portions is linked to the incorporation of the respective RPs into nascent ribosomal subunits is not understood, but it was shown to be important for ribosome functionality in yeast (Fernández-Pevida et al., 2016; Lacombe et al., 2009; Martín-Villanueva et al., 2020). In general, ubiquitin deconjugation is performed by a large family of deubiquitinases (DUBs) (Clague et al., 2019; Eletr and Wilkinson, 2014; Komander et al., 2009). About 100 DUBs exist in mammalian cells, subdivided into seven distinct families based on their structural organization (Clague et al., 2019; Komander et al., 2009). Biochemical experiments have identified several candidate DUBs for processing of the two human Ub-RP fusions, including UCHL3, USP9X, USP7, USP5 and OTULIN, which may act post-translationally in a partially redundant fashion (Grou et al., 2015).

Interestingly, humans and other holozoan organisms, a group not containing yeasts, synthesize a third RP as a fusion protein, namely eS30 (RPS30/FAU). Yet, in case of eS30, its N-terminal fusion partner is not ubiquitin but the ubiquitin-like protein (UBL) FUBI (also known as MNSFβ, FUB1, or UBIM). The coding sequence of FUBI-eS30 was originally identified in the transforming retrovirus FBR-MuSV (Finkel-Biskis-Reilly Murine Sarcoma Virus), which coined the term Fau (FBR-MuSV-associated ubiquitously expressed) for the FUBI-eS30 coding gene in mammals (Kas et al., 1992). FUBI displays 36% identity with ubiquitin. Similar to ubiquitin, FUBI possesses a C-terminal diglycine motif, which is crucial in ubiquitin for its conjugation to and removal from substrates including ubiquitin itself (Drag et al., 2008; Sloper-Mould et al., 2001). However, in contrast to ubiquitin, FUBI contains only a single internal lysine residue, corresponding to the least accessible lysine residue 27 of ubiquitin (Castañeda et al., 2016). Consistently, FUBI chain formation has not been reported, and enzymes mediating conjugation of FUBI to other proteins, akin to those that mediate protein ubiquitylation, remain to be identified (Cappadocia and Lima, 2018; van der Veen and Ploegh, 2012). Some studies suggested a role for FUBI in the immune system of mammals, that is in the regulation of T cells and macrophages. Such functions may entail the conjugation of FUBI to partner proteins such as the pro-apoptotic factor Bcl-G and the endocytosis regulator endophilin A2 (Nakamura et al., 2015; Nakamura and Shimosaki, 2009; Nakamura and Yamaguchi, 2006). However, since FUBI-eS30 is conserved in the holozoan subclade of Opisthokonta, which includes animals and their closest single-celled relatives, although not yeasts (Aleshin et al., 2007), the cellular immune system of multicellular organisms is unlikely to be the major determinant for the presence of FUBI.

Of the FUBI-eS30 fusion protein, only eS30 becomes a part of mature ribosomes. It is positioned at the shoulder of the small ribosomal subunit, tucked in between helices h16 and h18 of the 18S rRNA (Figure 1A). Three conserved basic residues near the C terminus of eS30 line the mRNA channel, where they may interact with the mRNA phosphate backbone (Rabl et al., 2011). The N-terminal end of eS30 extends towards the A-site decoding center that is located near the base of h44. Therefore, a FUBI moiety attached to the N terminus of eS30 may sterically obstruct the access of tRNAs to the ribosome. However, it has remained enigmatic which enzyme separates FUBI and eS30, and how FUBI cleavage is coordinated with the biogenesis of the 40S ribosomal subunit. Also, the exact consequences of a lack of FUBI removal on ribosome function or synthesis are currently unclear.

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Source data for Figure 1C and D with relevant bands labeled on the uncropped original blots.

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Here, we set out to address these open questions, exploiting uncleavable FUBI-eS30 constructs expressed in human cells. Persistence of FUBI on nascent 40S subunits impaired late cytoplasmic steps of 40S maturation, leading to the accumulation of subunits that fail to join the pool of translating ribosomes. Differential proteomics of factors associated with wild-type versus cleavage-deficient FUBI-eS30 mutants identified the nucleolar deubiquitinase USP36 as a candidate processing factor, which was proven by depletion and biochemical reconstitution experiments. Collectively, we demonstrate that removal of FUBI from eS30 is important for 40S subunits to gain competence in protein translation and show that USP36 is a promiscuous enzyme whose activity extends from ubiquitin to the ubiquitin-like protein FUBI.

To study the importance of FUBI-eS30 processing for ribosome biogenesis and function, we set out to explore the consequences of expressing cleavage-deficient FUBI-eS30 mutants. We reasoned that cleavage of the FUBI-eS30 fusion protein by an unidentified ubiquitin-like protease (ULP) could be impaired by mutation of FUBI’s C-terminal diglycine motif, akin to analogous mutations in ubiquitin that are known to affect endoproteolytic cleavage of linear ubiquitin fusion proteins (Bachmair et al., 1986; Butt et al., 1988; Lacombe et al., 2009; Martín-Villanueva et al., 2020). We generated tagged FUBI-eS30-StHA (tandem Strep II-hemagglutinin tag) wild-type (WT) or diglycine mutant constructs in which either both glycines were changed to alanines (G73,74A; AA) or the C-terminal glycine was changed to valine (G74V; GV) (Figure 1B), inspired by mutations previously introduced into Ub-RP fusion proteins in budding yeast (Lacombe et al., 2009). Using these constructs, we produced tetracycline-inducible HeLa and HEK cell lines in a cellular background containing endogenous FUBI-eS30 to uncouple the study of FUBI-eS30 processing from effects that could arise from lack of free FUBI or eS30. While most of FUBI(WT)-eS30-StHA was processed, alterations of the diglycine motif rendered both mutant constructs non-cleavable (Figure 1C). This indicates that a FUBI-eS30-specific ULP also requires an intact diglycine motif for processing.

To test whether the non-cleavable mutants can be incorporated into ribosomal subunits and translating ribosomes, we analyzed the sedimentation behavior of the WT and AA mutant constructs in sucrose gradients of lysates prepared from inducible HEK293 cell lines (Figure 1D). WT FUBI-eS30-StHA was cleaved and successfully incorporated into small ribosomal subunits that can engage in mRNA translation, evident by its co-sedimentation with the 40S-specific ribosome biogenesis factor (RBF) NOB1 in the 40S peak, and with the RPS uS3 in the 40S, 80S and polysome-containing fractions. In contrast, a larger portion of FUBI(AA)-eS30-StHA was migrating in lighter gradient fractions that contain monomeric proteins and small protein complexes, suggesting that its incorporation into 40S subunits could be less efficient. Still, FUBI(AA)-eS30-StHA was also detected in 40S- and 80S-containing fractions, indicating that the mutant fusion protein can in principle be incorporated into ribosomal subunits. Importantly, however, its levels were decreased in gradient fractions containing actively translating ribosomes and the polysome pellet. Thus, while FUBI removal is not essential for incorporation of eS30 into pre-40S particles, it appears to be a prerequisite for proper function of 40S subunits in translating polysomes.

To investigate the effects of non-cleavable FUBI-eS30-StHA mutant constructs on ribosomal subunit ratio and translation, we next performed polysome profile analysis of HEK293 cells expressing FUBI-eS30 constructs (Figure 2A). While there were no significant differences between the A₂₅₄ profiles of cell extracts obtained from parental HEK293 cells and cells expressing FUBI(WT)-eS30-StHA, expression of the non-cleavable FUBI-eS30-StHA AA and GV mutants led to significantly decreased 40S and polysome levels (Figure 2B). Concomitantly, free 60S subunit levels were increased, which is commonly observed if eukaryotic 40S production is impaired such as upon depletion of various RPS or 40S RBFs (Cheng et al., 2019; O'Donohue et al., 2010). Thus, defective maturation of pre-40S subunits containing mutant, non-cleavable FUBI-eS30 could be the reason why this fusion protein does not appear in polysomes (Figure 1D).

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Source data for Figure 2C with relevant areas labeled on the uncropped edited images.

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Unedited image of the ITS1 blot shown in Figure 2C.

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Unedited image of the ITS2 blot shown in Figure 2C.

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Unedited image of the gel shown in Figure 2C.

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eS30 is incorporated into nuclear pre-40S particles

In general, the timing of pre-40S incorporation of most RPS coincides with the major 40S maturation defects induced by their depletion (O'Donohue et al., 2010; Wild et al., 2010). However, for depletion of human eS30 (RPS30/FAU), different defects have been reported depending on the applied readout (O'Donohue et al., 2010; Wild et al., 2010). It has therefore remained difficult to deduce whether eS30 is incorporated into pre-40S subunits in the nucleus or in the cytoplasm. To test whether eS30 already joins nuclear pre-40S particles, we induced early 40S biogenesis defects by depletion of some key required factors (see below) and checked by immunofluorescence whether StHA-tagged eS30 derived from the FUBI(WT)-eS30-StHA fusion protein accumulates in the nucleus due to impaired subunit maturation (Figure 3A). To control for the efficiency of the respective treatments on 40S subunit maturation, we used the RBF ENP1 (BYSL) as a marker. ENP1 is a shuttling RBF that primarily localizes to nucleoli at steady state (si-control, -LMB) but accompanies maturing 40S subunits to the cytoplasm (Badertscher et al., 2015; Zemp et al., 2009). Upon treatment of parental HeLa or FUBI(WT)-eS30-StHA expressing cells with the XPO1 (CRM1)-specific nuclear export inhibitor leptomycin B (LMB) that blocks pre-40S export (Thomas and Kutay, 2003; Trotta et al., 2003), ENP1 accumulated in the nucleoplasm as expected (Figure 3A).

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Source data for Figure 3B with relevant areas labeled on the uncropped edited image of the gel and with relevant bands labeled on the uncropped original blots.

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Unedited image of the gel shown in Figure 3B.

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In untreated WT cells, eS30-StHA displayed a strong cytoplasmic signal, consistent with its presence in the active pool of ribosomal subunits (Figure 1D). Upon LMB treatment, a weak nucleoplasmic eS30-StHA signal became apparent in addition. Similarly, nuclear accumulation of eS30-StHA was also observed upon depletion of the pre-40S export receptor XPO1, suggesting that eS30-StHA is a part of nuclear pre-40S particles that accumulate in the nucleoplasm due to export inhibition. To exclude that this is simply due to the fact that eS30 itself is kept out of the nucleus by XPO1-mediated export, we directly interfered with nuclear 40S biogenesis by depletion of uS19 (RPS15), which is known to be required for late steps of nucleoplasmic pre-40S maturation (Léger-Silvestre et al., 2004; Rouquette et al., 2005). This led to a prominent nucleoplasmic accumulation of eS30-StHA, indicating that eS30 responds to perturbations of nuclear 40S maturation and is incorporated into nascent subunits in the nucleus. To examine whether eS30 might join pre-40S particles already in nucleoli, we depleted the primary binding RPS eS4X (Bernstein et al., 2004; Lastick, 1980; Wild et al., 2010), which is known to induce early nucleolar pre-40S subunit maturation defects (Badertscher et al., 2015; Wild et al., 2010). This resulted in the retention of ENP1 in nucleoli in presence of LMB as expected (Badertscher et al., 2015). Under these conditions, we observed both a nucleoplasmic and a nucleolar eS30-StHA signal, especially in presence of LMB, which could suggest that eS30 is targeted to nucleoli for its incorporation into pre-40S subunits. Collectively, the nucle(ol)ar accumulation of FUBI(WT)-eS30-StHA upon induction of nuclear 40S maturation and export defects suggests that eS30 assembles into nuclear pre-40S ribosomes, likely already in the nucleolus. This conclusion is supported by previous proteomic data that indicated an efficient accumulation of newly synthesized FUBI-eS30 in isolated nucleoli (Lam et al., 2007). To directly test whether eS30 is a component of nuclear pre-40S, we examined its association with pre-40S particles isolated by pull-down of a HASt-tagged version of the nucle(ol)ar RBF C21orf70 (FAM207A) from HEK293 cells (Zemp et al., 2014). Analysis of the pull-down eluates by immunoblotting revealed that eS30 is indeed associated with these nucle(ol)ar 40S precursors (Figure 3B) that also contained the early pre-40S RBF NOC4L, as expected (Zemp et al., 2014), and lacked eS26, which is incorporated into pre-40S subunits in the cytoplasm (Ameismeier et al., 2020; Plassart et al., 2021).

Non-cleavable FUBI-eS30 mutants induce defects in late cytoplasmic 40S subunit maturation

We next examined changes in the steady state localization of some key 40S RBFs upon expression of the non-cleavable FUBI-eS30 mutants, as this allows to more clearly pinpoint the step affected by a certain perturbation of 40S maturation. Nucleolar 40S subunit biogenesis gives rise to early pre-40S particles that contain a number of RBFs including ENP1, PNO1 (DIM2), and RRP12, all of which remain associated during transit of premature subunits from nucleoli through the nucleoplasm to the cytoplasm (Figure 4A). Further RBFs join in the nucleoplasm before nuclear export, including LTV1, TSR1, the atypical protein kinase RIOK2, and the site 3 endonuclease NOB1. Accordingly, there are various 40S RBFs that accompany maturing subunits from the nucleus to the cytoplasm, which are then successively released in coordination with structural rearrangements that shape the maturing pre-40S particles, supported by additional RBFs such as CK1, RIOK1, and EIF1AD (Ameismeier et al., 2018; Ameismeier et al., 2020; Mitterer et al., 2019; Plassart et al., 2021; Schäfer et al., 2006; Widmann et al., 2012; Zemp et al., 2014; Zemp et al., 2009).

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Immunofluorescence analysis of RIOK2, PNO1, NOB1, and RIOK1, which are among the last factors to be released during the final phase of 40S maturation in the cytoplasm, revealed strong changes in their localization and recycling behavior upon expression of the non-cleavable FUBI-eS30 mutants (Figure 4B). Strikingly, PNO1, an RBF that localizes to nucleoli under steady state conditions, relocated to the cytoplasm. Similarly, a prominent recycling defect was also evident for RIOK2 and NOB1. Both are cytoplasmic at steady state but in cells expressing non-cleavable FUBI-eS30-StHA, they failed to accumulate in the nucleus upon LMB treatment, reflecting a failure in their timely release from cytoplasmic pre-40S subunits (Zemp et al., 2014; Zemp et al., 2009). Also for RIOK1, which localizes to both the nucleus and the cytoplasm, cytoplasmic retention was clearly discernible. The observed changes are indicative for a late, cytoplasmic 40S biogenesis defect in cells expressing the non-cleavable FUBI-eS30 mutants, and consistent with the observed defects in 18S-E pre-rRNA processing (Figure 2C to F).

In contrast, those RBFs that are released at an earlier stage of cytoplasmic 40S maturation, namely LTV1, ENP1, and RRP12, remained largely unaffected by the expression of the non-cleavable FUBI-eS30 mutants (Figure 4—figure supplement 1A). LTV1, which is cytoplasmic at steady state, accumulated in the nucleoplasm of cells expressing either WT or mutant FUBI-eS30-StHA upon LMB treatment, demonstrating that its recycling is not perturbed by non-cleavable FUBI. For ENP1 and RRP12, two RBFs that predominantly localize to nucleoli and accumulate in the nucleoplasm upon LMB treatment, we observed a slight nucleolar retention in cells expressing the non-cleavable mutants. These changes together with the observed alterations in early pre-rRNA processing (Figure 2) could be the result of a so-called ‘rebound effect’, that is a secondary effect arising from the lack of 40S RBFs like PNO1 in nucleoli due to their strong cytoplasmic retention (Kofler et al., 2020; Zisser et al., 2018). Lastly, the localization of the 60S RBF NMD3 was not perturbed by mutant FUBI-eS30-StHA expression (Figure 4—figure supplement 1A), whereas depletion of the 60S RBF AAMP (Sqt1 in yeast) led to a cytoplasmic retention of NMD3 as expected (Figure 4—figure supplement 1B and C).

In summary, the comprehensive examination of the (re)localization of various RBFs suggests that FUBI-eS30 cleavage is a prerequisite for recycling of the RBFs RIOK2, PNO1, NOB1, and RIOK1 in final steps of cytoplasmic 40S maturation. In contrast, recycling of 40S RBFs that are released earlier as well as 60S biogenesis remain by-and-large unperturbed by the presence of FUBI in pre-40S particles.

Interestingly, when analyzing the localization of FUBI-eS30-StHA WT and its mutant derivatives in these experiments (Figure 4), we noticed a noteworthy difference: In contrast to the cytoplasmic staining of WT eS30-StHA, both non-cleavable mutants showed a reduced cytoplasmic signal and also localized to the nucleoplasm. This may reflect their hampered incorporation into maturing subunits since we observed a larger pool of non-cleavable FUBI-eS30 mutants in the light fractions of sucrose gradients compared to the mostly cleaved and 40S-associated WT (FUBI-)eS30-StHA (Figure 1D). As the partial nuclear accumulation of non-cleavable FUBI-eS30 mutants seemed at odds with the observed 40S maturation defects in the cytoplasm, we analyzed the fate of the nuclear FUBI-eS30 constructs by adding a chase period in tetracycline-free medium (Figure 4—figure supplement 2A). After the chase, the nuclear signal vanished and both mutants primarily localized to the cytoplasm like WT FUBI-eS30-StHA. Importantly, however, the late 40S subunit biogenesis defect persisted, as assessed by analysis of PNO1 localization (Figure 4—figure supplement 2B). Consistent with these data, immunoblot analysis of polysome profiles revealed that FUBI(AA)-eS30-StHA was mainly particle-associated after the chase period (Figure 4—figure supplement 2C and E). The disappearance of FUBI(AA)-eS30-StHA from lighter sucrose fractions could be due to its incorporation into pre-40S particles over time and/or degradation of the unincorporated protein, which may also be reflected by the generally weaker signal in immunofluorescence (Figure 4—figure supplement 2B). Importantly, since PNO1 was still trapped in the cytoplasm of AA- and GV-expressing cells and matching polysome profiles indicated 40S biogenesis defects similar to unchased cells, we conclude that the observed 40S biogenesis defects are primarily caused by pre-40S-associated non-cleavable FUBI-eS30-StHA mutants in the cytoplasm. Although we consider it unlikely, we cannot formally exclude that unincorporated, uncleaved FUBI-eS30 could serve as an unspecific ‘sink’ for one or some of the affected RBFs. However, at least NOB1 does not change its sedimentation behavior toward the pool of free protein in sucrose gradient analysis (Figure 4—figure supplement 2C and D), consistent with our conclusion.

Identification of FUBI-interacting candidate proteases

To characterize the molecular composition of FUBI-containing particles and to identify candidate FUBI-eS30 protease(s), we performed differential affinity purification mass spectrometry (AP-MS) experiments from lysates of HEK293 cells expressing either WT or non-cleavable FUBI-eS30-StHA constructs (Figure 5). We hypothesized that the non-cleavable mutants could serve as enzyme traps and enrich ULP(s), since ULP(s) may be able to bind to but not to cleave the mutant substrates, potentially hampering their release from the cleavage-resistant constructs.

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Source data for Figure 5A and C with relevant areas and bands labeled on the uncropped gel and uncropped original blots, respectively.

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Unedited image of the gel shown in Figure 5A.

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Comparison of the StrepTactin pull-down eluates of the HASt-GFP negative control with the WT and non-cleavable FUBI-eS30-StHA constructs by silver staining confirmed a clean enrichment of ribosomal particles based on the characteristic band patterns (Figure 5A, Montellese et al., 2020; Widmann et al., 2012; Wyler et al., 2011). The eluates of three independent experiments were subjected to MS analysis using data-dependent acquisition (Figure 5B, Supplementary file 1). In all (FUBI-)eS30-StHA pull-downs, ribosomal proteins of both subunits and a variety of 40S RBFs were the most strongly enriched factors (Supplementary file 1, Figure 5A and C). When inspecting the dataset for candidate FUBI processing enzymes, we found three DUBs to confidently interact with the FUBI-eS30 constructs, namely USP16, USP10, and USP36, all of which belong to the ubiquitin-specific protease (USP) family. While USP16, an established 40S RBF known to remove ubiquitin from eS31 (Rps27a) (Montellese et al., 2020), was equally co-purified with both WT and non-cleavable FUBI-eS30-StHA baits, USP10 and USP36 were enriched on the non-cleavable mutants (Figure 5B). Importantly, these observations were confirmed by immunoblotting of the pull-down eluates (Figure 5C), which made USP10 and USP36 prime ULP candidates for FUBI-eS30 processing.

USP10 is a highly conserved cytoplasmic DUB and has been previously described to interact with 40S subunits (Clague et al., 2019; Kapadia and Gartenhaus, 2019). It is involved in resolution of stalled translation elongation complexes through removal of the regulatory monoubiquitylations from eS10, uS3, and uS5 (Garshott et al., 2020; Jung et al., 2017; Meyer et al., 2020). In contrast, USP36 is the only known catalytically active DUB exclusively localizing to human nucleoli (Clague et al., 2019; Endo et al., 2009a; Scherl et al., 2002). It has been implicated in the deubiquitylation of various nucleolar proteins, including the catalytic subunit of RNA polymerase I, RPA194 (Rpa190 in yeast; Peltonen et al., 2014; Richardson et al., 2012), the helicase DHX33 (Dhr2 in yeast; Fraile et al., 2018), the 2’-O-methyltransferase FBL, and nucleophosmin (NPM/B23, Endo et al., 2009a). Interestingly, USP36-mediated deubiquitylation has also been shown to lead to the stabilization of the transcription factor Myc (Sun et al., 2015; Thevenon et al., 2020), a proto-oncogenic regulator of ribosome and protein synthesis (van Riggelen et al., 2010).

Besides the two FUBI-specific candidate proteases, we found several additional proteins enriched in the eluates of pull-downs with the non-cleavable mutants (Figure 5B, Supplementary file 1). Surprisingly, a number of 60S RBFs (Fatica and Tollervey, 2002; Nissan et al., 2002; Woolford and Baserga, 2013), including NMD3, LSG1, GNL3L, and GNL2, were significantly enriched on the mutants (Figure 5B and C), indicating that the pre-40S subunits arrested in their maturation by the presence of FUBI may associate with pre-60S subunits. Immunoblotting indeed confirmed the enrichment of NMD3 and LSG1. Whether this enrichment represents a biologically meaningful interaction, is aberrantly induced by the stalled particles or caused by post-lysis association cannot be deduced from this data.

Among the identified RBFs of the 40S subunit, the protein kinase RIOK1, which is usually harder to capture on pre-40S subunits than other 40S RBFs (Widmann et al., 2012), was clearly enriched on the non-cleavable mutants (Figure 5C), consistent with our recycling data (Figure 4). Interestingly, we also observed a shift in migration of RIOK2 induced by the expression of the non-cleavable FUBI-eS30 constructs, potentially caused by the arrest of an activated, phosphorylated form of the kinase on pre-40S subunits. Another observation was the striking scarcity of peptides of the recently identified 40S RBF EIF1AD (Ameismeier et al., 2020) in the MS data, and it was also barely detectable in the eluates by immunoblotting (Supplementary file 1, Figure 5C).

USP36 promotes FUBI-eS30 processing in vivo

We next analyzed whether the identified candidate proteases USP10 or USP36 contribute to FUBI-eS30 processing in living cells. Both DUBs were depleted from HeLa cells by RNAi and cell extracts were analyzed by immunoblotting for accumulation of uncleaved endogenous FUBI-eS30 using an antibody raised against FUBI (Figure 6A). Quantification of the level of uncleaved FUBI-eS30 revealed no change upon depletion of USP10 using four different siRNAs. In contrast, the amount of uncleaved FUBI-eS30 was significantly elevated upon treatment of the cells with three of the four USP36-targeting siRNAs. The extent of accumulation of uncleaved FUBI-eS30 varied between the USP36 siRNAs despite similar levels of downregulation. This could either be due to different kinetics of downregulation for the individual siRNAs or to off-target effects altering cellular processes or factors that further attenuate or promote FUBI-eS30 processing.

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To ensure that both parts of the FAU fusion protein, that is both FUBI and eS30, jointly accumulate as the assigned uncleaved protein species in form of an in cis fusion upon USP36 depletion, HEK293 cell lines expressing either N- or C-terminally tagged FUBI-eS30 were treated with control or USP36 siRNA (Figure 6B). Upon depletion of USP36, uncleaved tagged FUBI-eS30 accumulated in both cell lines as demonstrated by immunoblotting using an antibody against the HA tag. Collectively, these results indicate that separation of FUBI and eS30 depends on USP36 in vivo.

To further strengthen this conclusion by an independent method, we employed a complementary CRISPR interference (CRISPRi) approach (Boneberg et al., 2019). We used two different guide RNA sequences to target a gene-silencing, catalytically inactive KRAB-dCas9 protein to the promoter region of USP36 (Gilbert et al., 2013; Horlbeck et al., 2016; Qi et al., 2013). With both guides, USP36 expression was efficiently downregulated, again leading to a significant accumulation of uncleaved endogenous FUBI-eS30 (Figure 6C). Importantly, this could be rescued in cell lines expressing EGFP-tagged WT USP36 but not a catalytically inactive mutant, in which the active site cysteine was mutated to an alanine (C131A, CA; Figure 6C). In these experiments, we also noted a slight dominant-negative effect of expression of the catalytically inactive USP36 mutant on FUBI-eS30 processing. In conclusion, the CRISPRi-rescue experiment showed that USP36 and its catalytic activity are required for FUBI-eS30 processing.

USP36 cleaves FUBI fusion proteins in vitro

Since the catalytic activity of USP36 is required for FUBI-eS30 processing in vivo, we finally wished to explore whether USP36 can indeed mediate the cleavage of FUBI-containing fusion proteins in vitro. We expressed both full-length USP36 WT and the CA mutant in insect cells and purified the proteins by StrepTactin affinity purification. Then, we performed in vitro processing assays using purified His₆-tagged FUBI(WT)-eS30 or the cleavage-resistant FUBI(AA)-eS30 mutant as substrates sampling different time points during the reaction (Figure 7A and B). His₆-FUBI(WT)-eS30 was indeed successfully processed into FUBI and eS30 by wild-type USP36. The cleavage products were already apparent at the first time point of 7.5 min and about half of the substrate was turned over after about 20 min at the chosen conditions (Figure 7A and E). In contrast, the inactive USP36 CA mutant did not promote processing, excluding that the observed FUBI(WT)-eS30 cleavage was due to any co-purifying activity in the enzyme preparation. As expected, the non-cleavable FUBI(AA)-eS30 mutant was resistant to attack by WT USP36 (Figure 7B). Together, this data provides direct evidence that USP36 can act as a ULP for FUBI-eS30.

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USP36 has been classified as a DUB and proposed to deubiquitylate several nucle(ol)ar proteins, thereby preventing their proteasomal degradation (Endo et al., 2009a; Fraile et al., 2018; Sun et al., 2015). With respect to its capacity to process linear ubiquitin fusions, a USP36 fragment encompassing its catalytic domain has been reported to cleave di-ubiquitin substrates in vitro, albeit inefficiently (Ritorto et al., 2014). We therefore directly compared the processing activity of USP36 toward FUBI-EGFP and Ub-EGFP fusion proteins (Figure 7C and D). Interestingly, both FUBI- and Ub-EGFP were cleaved by USP36 WT but not the CA mutant. Cleavage of FUBI- and Ub-EGFP occurred with by-and-large comparable kinetics (Figure 7E), indicating that FUBI is not a preferred substrate in the context of the EGFP fusion proteins. In conclusion, the DUB USP36 is a promiscuous enzyme that can process both linear FUBI and ubiquitin fusion proteins in vitro.

In this work, we have investigated how processing of the exceptional ribosomal protein eS30 that is synthesized as a fusion with the ubiquitin-like protein FUBI, is coordinated with the biogenesis of 40S ribosomal subunits. Our data indicate that eS30 joins pre-40S subunits in the nucleus, where FUBI is cut off from eS30 by the DUB USP36. Non-cleavable mutants of FUBI-eS30 can be incorporated into pre-40S particles, albeit less efficiently than WT, and exert a dominant-negative effect on pre-rRNA processing and recycling of RBFs in the cytoplasm. 40S subunits carrying a persistent FUBI moiety are impaired in entering the pool of translating ribosomes, demonstrating that FUBI processing is an important aspect of 40S subunit maturation.

A list of materials is provided in Appendix key resources table.

Source data have been provided for Figure 5.

1. 1. Kutay U

   (2020) PRIDE

   ID PXD021864. AP-MS analysis of human FUBIeS30/FAU WT and non-cleavable G73,4A and G74V mutants.

   https://www.ebi.ac.uk/pride/archive/projects/PXD021864

1. 1. Aleshin VV
   2. Konstantinova AV
   3. Mikhailov KV
   4. Nikitin MA
   5. Petrov NB

   (2007) Do we need many genes for phylogenetic inference?

   Biochemistry. Biokhimiia 72:1313–1323.

   https://doi.org/10.1134/s000629790712005x

   • PubMed
   • Google Scholar
2. 1. Ameismeier M
   2. Cheng J
   3. Berninghausen O
   4. Beckmann R

   (2018) Visualizing late states of human 40S ribosomal subunit maturation

   Nature 558:249–253.

   https://doi.org/10.1038/s41586-018-0193-0

   • PubMed
   • Google Scholar
3. 1. Ameismeier M
   2. Zemp I
   3. van den Heuvel J
   4. Thoms M
   5. Berninghausen O
   6. Kutay U
   7. Beckmann R

   (2020) Structural basis for the final steps of human 40S ribosome maturation

   Nature 587:683–687.

   https://doi.org/10.1038/s41586-020-2929-x

   • PubMed
   • Google Scholar
4. 1. Angermayr M
   2. Hochleitner E
   3. Lottspeich F
   4. Bandlow W

   (2007) Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast

   The FEBS Journal 274:4654–4667.

   https://doi.org/10.1111/j.1742-4658.2007.05993.x

   • PubMed
   • Google Scholar
5. 1. Bachmair A
   2. Finley D
   3. Varshavsky A

   (1986) In vivo half-life of a protein is a function of its amino-terminal residue

   Science 234:179–186.

   https://doi.org/10.1126/science.3018930

   • PubMed
   • Google Scholar
6. 1. Badertscher L
   2. Wild T
   3. Montellese C
   4. Alexander LT
   5. Bammert L
   6. Sarazova M
   7. Stebler M
   8. Csucs G
   9. Mayer TU
   10. Zamboni N
   11. Zemp I
   12. Horvath P
   13. Kutay U

   (2015) Genome-wide RNAi screening identifies protein modules required for 40S subunit synthesis in human cells

   Cell Reports 13:2879–2891.

   https://doi.org/10.1016/j.celrep.2015.11.061

   • PubMed
   • Google Scholar
7. 1. Baker RT
   2. Board PG

   (1991) The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes

   Nucleic Acids Research 19:1035–1040.

   https://doi.org/10.1093/nar/19.5.1035

   • PubMed
   • Google Scholar
8. 1. Bernstein KA
   2. Gallagher JE
   3. Mitchell BM
   4. Granneman S
   5. Baserga SJ

   (2004) The small-subunit processome is a ribosome assembly intermediate

   Eukaryotic Cell 3:1619–1626.

   https://doi.org/10.1128/EC.3.6.1619-1626.2004

   • PubMed
   • Google Scholar
9. 1. Bianchi M
   2. Giacomini E
   3. Crinelli R
   4. Radici L
   5. Carloni E
   6. Magnani M

   (2015) Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants

   Gene 573:100–109.

   https://doi.org/10.1016/j.gene.2015.07.030

   • PubMed
   • Google Scholar
10. 1. Boneberg FM
    2. Brandmann T
    3. Kobel L
    4. van den Heuvel J
    5. Bargsten K
    6. Bammert L
    7. Kutay U
    8. Jinek M

    (2019) Molecular mechanism of the RNA helicase DHX37 and its activation by UTP14A in ribosome biogenesis

    RNA 25:685–701.

    https://doi.org/10.1261/rna.069609.118

    • PubMed
    • Google Scholar
11. 1. Butt TR
    2. Khan MI
    3. Marsh J
    4. Ecker DJ
    5. Crooke ST

    (1988) Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions

    Journal of Biological Chemistry 263:16364–16371.

    https://doi.org/10.1016/S0021-9258(18)37602-6

    • PubMed
    • Google Scholar
12. 1. Cappadocia L
    2. Lima CD

    (2018) Ubiquitin-like protein conjugation: structures, chemistry, and mechanism

    Chemical Reviews 118:889–918.

    https://doi.org/10.1021/acs.chemrev.6b00737

    • PubMed
    • Google Scholar
13. 1. Castañeda CA
    2. Dixon EK
    3. Walker O
    4. Chaturvedi A
    5. Nakasone MA
    6. Curtis JE
    7. Reed MR
    8. Krueger S
    9. Cropp TA
    10. Fushman D

    (2016) Linkage via K27 bestows ubiquitin chains with unique properties among polyubiquitins

    Structure 24:423–436.

    https://doi.org/10.1016/j.str.2016.01.007

    • PubMed
    • Google Scholar
14. 1. Cheng Z
    2. Mugler CF
    3. Keskin A
    4. Hodapp S
    5. Chan LY
    6. Weis K
    7. Mertins P
    8. Regev A
    9. Jovanovic M
    10. Brar GA

    (2019) Small and large ribosomal subunit deficiencies lead to distinct gene expression signatures that reflect cellular growth rate

    Molecular Cell 73:36–47.

    https://doi.org/10.1016/j.molcel.2018.10.032

    • PubMed
    • Google Scholar
15. 1. Clague MJ
    2. Urbé S
    3. Komander D

    (2019) Breaking the chains: deubiquitylating enzyme specificity begets function

    Nature Reviews. Molecular Cell Biology 20:338–352.

    https://doi.org/10.1038/s41580-019-0099-1

    • PubMed
    • Google Scholar
16. 1. Drag M
    2. Mikolajczyk J
    3. Bekes M
    4. Reyes-Turcu FE
    5. Ellman JA
    6. Wilkinson KD
    7. Salvesen GS

    (2008) Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes)

    The Biochemical Journal 415:367–375.

    https://doi.org/10.1042/BJ20080779

    • PubMed
    • Google Scholar
17. 1. Eletr ZM
    2. Wilkinson KD

    (2014) Regulation of proteolysis by human deubiquitinating enzymes

    Biochimica et Biophysica Acta 1843:114–128.

    https://doi.org/10.1016/j.bbamcr.2013.06.027

    • PubMed
    • Google Scholar
18. 1. Endo A
    2. Kitamura N
    3. Komada M

    (2009a) Nucleophosmin/B23 regulates ubiquitin dynamics in nucleoli by recruiting deubiquitylating enzyme USP36

    Journal of Biological Chemistry 284:27918–27923.

    https://doi.org/10.1074/jbc.M109.037218

    • Google Scholar
19. 1. Endo A
    2. Matsumoto M
    3. Inada T
    4. Yamamoto A
    5. Nakayama KI
    6. Kitamura N
    7. Komada M

    (2009b) Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36

    Journal of Cell Science 122:678–686.

    https://doi.org/10.1242/jcs.044461

    • PubMed
    • Google Scholar
20. 1. Fatica A
    2. Oeffinger M
    3. Dlakić M
    4. Tollervey D

    (2003) Nob1p is required for cleavage of the 3' end of 18S rRNA

    Molecular and Cellular Biology 23:1798–1807.

    https://doi.org/10.1128/MCB.23.5.1798-1807.2003

    • PubMed
    • Google Scholar
21. 1. Fatica A
    2. Tollervey D

    (2002) Making ribosomes

    Current Opinion in Cell Biology 14:313–318.

    https://doi.org/10.1016/s0955-0674(02)00336-8

    • PubMed
    • Google Scholar
22. 1. Fernández-Pevida A
    2. Martín-Villanueva S
    3. Murat G
    4. Lacombe T
    5. Kressler D
    6. de la Cruz J

    (2016) The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

    Nucleic Acids Research 44:7777–7791.

    https://doi.org/10.1093/nar/gkw641

    • PubMed
    • Google Scholar
23. 1. Ferreira-Cerca S
    2. Kiburu I
    3. Thomson E
    4. LaRonde N
    5. Hurt E

    (2014) Dominant Rio1 kinase/ATPase catalytic mutant induces trapping of late pre-40S biogenesis factors in 80S-like ribosomes

    Nucleic Acids Research 42:8635–8647.

    https://doi.org/10.1093/nar/gku542

    • PubMed
    • Google Scholar
24. 1. Finley D
    2. Ozkaynak E
    3. Varshavsky A

    (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses

    Cell 48:1035–1046.

    https://doi.org/10.1016/0092-8674(87)90711-2

    • PubMed
    • Google Scholar
25. 1. Finley D
    2. Bartel B
    3. Varshavsky A

    (1989) The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis

    Nature 338:394–401.

    https://doi.org/10.1038/338394a0

    • PubMed
    • Google Scholar
26. 1. Fraile JM
    2. Campos-Iglesias D
    3. Rodríguez F
    4. Astudillo A
    5. Vilarrasa-Blasi R
    6. Verdaguer-Dot N
    7. Prado MA
    8. Paulo JA
    9. Gygi SP
    10. Martín-Subero JI
    11. Freije JMP
    12. López-Otín C

    (2018) Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice

    The Journal of Biological Chemistry 293:2183–2194.

    https://doi.org/10.1074/jbc.M117.788430

    • PubMed
    • Google Scholar
27. 1. Garshott DM
    2. Sundaramoorthy E
    3. Leonard M
    4. Bennett EJ

    (2020) Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized

    eLife 9:e54023.

    https://doi.org/10.7554/eLife.54023

    • PubMed
    • Google Scholar
28. 1. Geerlings TH
    2. Faber AW
    3. Bister MD
    4. Vos JC
    5. Raué HA

    (2003) Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20 S Pre-rRNA in Saccharomyces cerevisiae

    The Journal of Biological Chemistry 278:22537–22545.

    https://doi.org/10.1074/jbc.M300759200

    • PubMed
    • Google Scholar
29. 1. Gilbert LA
    2. Larson MH
    3. Morsut L
    4. Liu Z
    5. Brar GA
    6. Torres SE
    7. Stern-Ginossar N
    8. Brandman O
    9. Whitehead EH
    10. Doudna JA
    11. Lim WA
    12. Weissman JS
    13. Qi LS

    (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes

    Cell 154:442–451.

    https://doi.org/10.1016/j.cell.2013.06.044

    • PubMed
    • Google Scholar
30. 1. Grou CP
    2. Pinto MP
    3. Mendes AV
    4. Domingues P
    5. Azevedo JE

    (2015) The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors

    Scientific Reports 5:12836.

    https://doi.org/10.1038/srep12836

    • PubMed
    • Google Scholar
31. 1. Häfner J
    2. Mayr MI
    3. Möckel MM
    4. Mayer TU

    (2014) Pre-anaphase chromosome oscillations are regulated by the antagonistic activities of Cdk1 and PP1 on Kif18A

    Nature Communications 5:4397.

    https://doi.org/10.1038/ncomms5397

    • PubMed
    • Google Scholar
32. 1. Henras AK
    2. Plisson-Chastang C
    3. O'Donohue MF
    4. Chakraborty A
    5. Gleizes PE

    (2015) An overview of pre-ribosomal RNA processing in eukaryotes

    Wiley interdisciplinary reviews. RNA 6:225–242.

    https://doi.org/10.1002/wrna.1269

    • PubMed
    • Google Scholar
33. 1. Heuer A
    2. Thomson E
    3. Schmidt C
    4. Berninghausen O
    5. Becker T
    6. Hurt E
    7. Beckmann R

    (2017) Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae

    eLife 6:e30189.

    https://doi.org/10.7554/eLife.30189

    • PubMed
    • Google Scholar
34. 1. Horlbeck MA
    2. Gilbert LA
    3. Villalta JE
    4. Adamson B
    5. Pak RA
    6. Chen Y
    7. Fields AP
    8. Park CY
    9. Corn JE
    10. Kampmann M
    11. Weissman JS

    (2016) Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    eLife 5:e19760.

    https://doi.org/10.7554/eLife.19760

    • PubMed
    • Google Scholar
35. 1. Johnson MC
    2. Ghalei H
    3. Doxtader KA
    4. Karbstein K
    5. Stroupe ME

    (2017) Structural heterogeneity in Pre-40S ribosomes

    Structure 25:329–340.

    https://doi.org/10.1016/j.str.2016.12.011

    • PubMed
    • Google Scholar
36. 1. Jung Y
    2. Kim HD
    3. Yang HW
    4. Kim HJ
    5. Jang CY
    6. Kim J

    (2017) Modulating cellular balance of Rps3 mono-ubiquitination by both Hel2 E3 ligase and Ubp3 deubiquitinase regulates protein quality control

    Experimental & Molecular Medicine 49:e390.

    https://doi.org/10.1038/emm.2017.128

    • PubMed
    • Google Scholar
37. 1. Kapadia BB
    2. Gartenhaus RB

    (2019) DUBbing down translation: the functional interaction of deubiquitinases with the translational machinery

    Molecular Cancer Therapeutics 18:1475–1483.

    https://doi.org/10.1158/1535-7163.MCT-19-0307

    • PubMed
    • Google Scholar
38. 1. Kas K
    2. Michiels L
    3. Merregaert J

    (1992) Genomic structure and expression of the human fau gene: encoding the ribosomal protein S30 fused to a ubiquitin-like protein

    Biochemical and Biophysical Research Communications 187:927–933.

    https://doi.org/10.1016/0006-291x(92)91286-y

    • PubMed
    • Google Scholar
39. 1. Khatter H
    2. Myasnikov AG
    3. Natchiar SK
    4. Klaholz BP

    (2015) Structure of the human 80S ribosome

    Nature 520:640–645.

    https://doi.org/10.1038/nature14427

    • PubMed
    • Google Scholar
40. 1. Kirschner LS
    2. Stratakis CA

    (2000) Structure of the human ubiquitin fusion gene Uba80 (RPS27a) and one of its pseudogenes

    Biochemical and Biophysical Research Communications 270:1106–1110.

    https://doi.org/10.1006/bbrc.2000.2568

    • PubMed
    • Google Scholar
41. 1. Kofler L
    2. Prattes M
    3. Bergler H

    (2020) From snapshots to Flipbook—Resolving the Dynamics of Ribosome Biogenesis with Chemical Probes

    International Journal of Molecular Sciences 21:2998.

    https://doi.org/10.3390/ijms21082998

    • Google Scholar
42. 1. Komander D
    2. Clague MJ
    3. Urbé S

    (2009) Breaking the chains: structure and function of the deubiquitinases

    Nature Reviews. Molecular Cell Biology 10:550–563.

    https://doi.org/10.1038/nrm2731

    • PubMed
    • Google Scholar
43. 1. Lacombe T
    2. García-Gómez JJ
    3. de la Cruz J
    4. Roser D
    5. Hurt E
    6. Linder P
    7. Kressler D

    (2009) Linear ubiquitin fusion to Rps31 and its subsequent cleavage are required for the efficient production and functional integrity of 40S ribosomal subunits

    Molecular Microbiology 72:69–84.

    https://doi.org/10.1111/j.1365-2958.2009.06622.x

    • PubMed
    • Google Scholar
44. 1. Lam YW
    2. Lamond AI
    3. Mann M
    4. Andersen JS

    (2007) Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins

    Current Biology: CB 17:749–760.

    https://doi.org/10.1016/j.cub.2007.03.064

    • PubMed
    • Google Scholar
45. 1. Larburu N
    2. Montellese C
    3. O'Donohue MF
    4. Kutay U
    5. Gleizes PE
    6. Plisson-Chastang C

    (2016) Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

    Nucleic Acids Research 44:8465–8478.

    https://doi.org/10.1093/nar/gkw714

    • PubMed
    • Google Scholar
46. 1. Larsen CN
    2. Krantz BA
    3. Wilkinson KD

    (1998) Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases

    Biochemistry 37:3358–3368.

    https://doi.org/10.1021/bi972274d

    • PubMed
    • Google Scholar
47. 1. Lastick SM

    (1980) The assembly of ribosomes in HeLa cell nucleoli

    European Journal of Biochemistry 113:175–182.

    https://doi.org/10.1111/j.1432-1033.1980.tb06152.x

    • PubMed
    • Google Scholar
48. 1. Lebaron S
    2. Schneider C
    3. van Nues RW
    4. Swiatkowska A
    5. Walsh D
    6. Böttcher B
    7. Granneman S
    8. Watkins NJ
    9. Tollervey D

    (2012) Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

    Nature Structural & Molecular Biology 19:744–753.

    https://doi.org/10.1038/nsmb.2308

    • PubMed
    • Google Scholar
49. 1. Léger-Silvestre I
    2. Milkereit P
    3. Ferreira-Cerca S
    4. Saveanu C
    5. Rousselle JC
    6. Choesmel V
    7. Guinefoleau C
    8. Gas N
    9. Gleizes PE

    (2004) The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

    The EMBO journal 23:2336–2347.

    https://doi.org/10.1038/sj.emboj.7600252

    • PubMed
    • Google Scholar
50. 1. Lemak A

    (2011)

    2l7r: solution NMR structure of N-terminal Ubiquitin-like domain of FUBI, a ribosomal protein S30 precursor from Homo sapiens. NorthEast structural genomics consortium (NESG) target HR6166

    PDB.

    • Google Scholar
51. 1. Lund PK
    2. Moats-Staats BM
    3. Simmons JG
    4. Hoyt E
    5. D'Ercole AJ
    6. Martin F
    7. Van Wyk JJ

    (1985)

    Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor

    The Journal of Biological Chemistry 260:7609–7613.

    • PubMed
    • Google Scholar
52. 1. Martín-Villanueva S
    2. Fernández-Pevida A
    3. Kressler D
    4. de la Cruz J

    (2019) The ubiquitin moiety of Ubi1 is required for productive expression of ribosomal protein eL40 in Saccharomyces cerevisiae

    Cells 8:850.

    https://doi.org/10.3390/cells8080850

    • Google Scholar
53. 1. Martín-Villanueva S
    2. Fernández-Pevida A
    3. Fernández-Fernández J
    4. Kressler D
    5. de la Cruz J

    (2020) Ubiquitin release from eL40 is required for cytoplasmic maturation and function of 60S ribosomal subunits in Saccharomyces cerevisiae

    The FEBS Journal 287:345–360.

    https://doi.org/10.1111/febs.14999

    • PubMed
    • Google Scholar
54. 1. Martín-Villanueva S
    2. Gutiérrez G
    3. Kressler D
    4. de la Cruz J

    (2021) Ubiquitin and Ubiquitin-Like proteins and domains in ribosome production and function: chance or necessity?

    International Journal of Molecular Sciences 22:4359.

    https://doi.org/10.3390/ijms22094359

    • PubMed
    • Google Scholar
55. 1. McQuin C
    2. Goodman A
    3. Chernyshev V
    4. Kamentsky L
    5. Cimini BA
    6. Karhohs KW
    7. Doan M
    8. Ding L
    9. Rafelski SM
    10. Thirstrup D
    11. Wiegraebe W
    12. Singh S
    13. Becker T
    14. Caicedo JC
    15. Carpenter AE

    (2018) CellProfiler 3.0: Next-generation image processing for biology

    PLOS Biology 16:e2005970.

    https://doi.org/10.1371/journal.pbio.2005970

    • PubMed
    • Google Scholar
56. 1. Mevissen TET
    2. Komander D

    (2017) Mechanisms of Deubiquitinase Specificity and Regulation

    Annual Review of Biochemistry 86:159–192.

    https://doi.org/10.1146/annurev-biochem-061516-044916

    • PubMed
    • Google Scholar
57. 1. Meyer C
    2. Garzia A
    3. Morozov P
    4. Molina H
    5. Tuschl T

    (2020) The G3BP1-Family-USP10 deubiquitinase complex rescues ubiquitinated 40S subunits of ribosomes stalled in translation from lysosomal degradation

    Molecular Cell 77:1193–1205.

    https://doi.org/10.1016/j.molcel.2019.12.024

    • Google Scholar
58. 1. Mitterer V
    2. Shayan R
    3. Ferreira-Cerca S
    4. Murat G
    5. Enne T
    6. Rinaldi D
    7. Weigl S
    8. Omanic H
    9. Gleizes PE
    10. Kressler D
    11. Plisson-Chastang C
    12. Pertschy B

    (2019) Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism

    Nature Communications 10:2754.

    https://doi.org/10.1038/s41467-019-10678-z

    • PubMed
    • Google Scholar
59. 1. Montellese C
    2. van den Heuvel J
    3. Ashiono C
    4. Dörner K
    5. Melnik A
    6. Jonas S
    7. Zemp I
    8. Picotti P
    9. Gillet LC
    10. Kutay U

    (2020) USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit

    eLife 9:e54435.

    https://doi.org/10.7554/eLife.54435

    • PubMed
    • Google Scholar
60. 1. Nakamura M
    2. Nakagawa M
    3. Watanabe J

    (2015) Ubiquitin-like protein MNSFβ negatively regulates T cell function and survival

    Immunological Investigations 44:1–12.

    https://doi.org/10.3109/08820139.2014.909454

    • PubMed
    • Google Scholar
61. 1. Nakamura M
    2. Shimosaki S

    (2009) The ubiquitin-like protein monoclonal nonspecific suppressor factor beta conjugates to endophilin II and regulates phagocytosis

    The FEBS Journal 276:6355–6363.

    https://doi.org/10.1111/j.1742-4658.2009.07348.x

    • PubMed
    • Google Scholar
62. 1. Nakamura M
    2. Yamaguchi S

    (2006) The ubiquitin-like protein MNSFbeta regulates ERK-MAPK cascade

    The Journal of Biological Chemistry 281:16861–16869.

    https://doi.org/10.1074/jbc.M509907200

    • PubMed
    • Google Scholar
63. 1. Nissan TA
    2. Bassler J
    3. Petfalski E
    4. Tollervey D
    5. Hurt E

    (2002) 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm

    The EMBO Journal 21:5539–5547.

    https://doi.org/10.1093/emboj/cdf547

    • PubMed
    • Google Scholar
64. 1. O'Donohue MF
    2. Choesmel V
    3. Faubladier M
    4. Fichant G
    5. Gleizes PE

    (2010) Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits

    The Journal of Cell Biology 190:853–866.

    https://doi.org/10.1083/jcb.201005117

    • PubMed
    • Google Scholar
65. 1. Ozkaynak E
    2. Finley D
    3. Solomon MJ
    4. Varshavsky A

    (1987) The yeast ubiquitin genes: a family of natural gene fusions

    The EMBO Journal 6:1429–1439.

    https://doi.org/10.1002/j.1460-2075.1987.tb02384.x

    • Google Scholar
66. 1. Peltonen K
    2. Colis L
    3. Liu H
    4. Jäämaa S
    5. Zhang Z
    6. Af Hällström T
    7. Moore HM
    8. Sirajuddin P
    9. Laiho M

    (2014) Small molecule BMH-compounds that inhibit RNA polymerase I and cause nucleolar stress

    Molecular Cancer Therapeutics 13:2537–2546.

    https://doi.org/10.1158/1535-7163.MCT-14-0256

    • PubMed
    • Google Scholar
67. 1. Pertschy B
    2. Schneider C
    3. Gnädig M
    4. Schäfer T
    5. Tollervey D
    6. Hurt E

    (2009) RNA helicase Prp43 and its co-factor Pfa1 promote 20 to 18 S rRNA processing catalyzed by the endonuclease Nob1

    The Journal of Biological Chemistry 284:35079–35091.

    https://doi.org/10.1074/jbc.M109.040774

    • PubMed
    • Google Scholar
68. 1. Plassart L
    2. Shayan R
    3. Montellese C
    4. Rinaldi D
    5. Larburu N
    6. Pichereaux C
    7. Froment C
    8. Lebaron S
    9. O'Donohue MF
    10. Kutay U
    11. Marcoux J
    12. Gleizes PE
    13. Plisson-Chastang C

    (2021) The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

    eLife 10:e61254.

    https://doi.org/10.7554/eLife.61254

    • PubMed
    • Google Scholar
69. 1. Preti M
    2. O'Donohue MF
    3. Montel-Lehry N
    4. Bortolin-Cavaillé ML
    5. Choesmel V
    6. Gleizes PE

    (2013) Gradual processing of the ITS1 from the nucleolus to the cytoplasm during synthesis of the human 18S rRNA

    Nucleic Acids Research 41:4709–4723.

    https://doi.org/10.1093/nar/gkt160

    • PubMed
    • Google Scholar
70. 1. Qi LS
    2. Larson MH
    3. Gilbert LA
    4. Doudna JA
    5. Weissman JS
    6. Arkin AP
    7. Lim WA

    (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression

    Cell 152:1173–1183.

    https://doi.org/10.1016/j.cell.2013.02.022

    • PubMed
    • Google Scholar
71. 1. Rabl J
    2. Leibundgut M
    3. Ataide SF
    4. Haag A
    5. Ban N

    (2011) Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1

    Science 331:730–736.

    https://doi.org/10.1126/science.1198308

    • PubMed
    • Google Scholar
72. 1. Richardson LA
    2. Reed BJ
    3. Charette JM
    4. Freed EF
    5. Fredrickson EK
    6. Locke MN
    7. Baserga SJ
    8. Gardner RG

    (2012) A conserved deubiquitinating enzyme controls cell growth by regulating RNA polymerase I stability

    Cell Reports 2:372–385.

    https://doi.org/10.1016/j.celrep.2012.07.009

    • PubMed
    • Google Scholar
73. 1. Ritorto MS
    2. Ewan R
    3. Perez-Oliva AB
    4. Knebel A
    5. Buhrlage SJ
    6. Wightman M
    7. Kelly SM
    8. Wood NT
    9. Virdee S
    10. Gray NS
    11. Morrice NA
    12. Alessi DR
    13. Trost M

    (2014) Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

    Nature Communications 5:.

    https://doi.org/10.1038/ncomms5763

    • Google Scholar
74. 1. Rouquette J
    2. Choesmel V
    3. Gleizes PE

    (2005) Nuclear export and cytoplasmic processing of precursors to the 40S ribosomal subunits in mammalian cells

    The EMBO Journal 24:2862–2872.

    https://doi.org/10.1038/sj.emboj.7600752

    • PubMed
    • Google Scholar
75. 1. Ryu H
    2. Sun XX
    3. Chen Y
    4. Li Y
    5. Wang X
    6. Dai RS
    7. Zhu HM
    8. Klimek J
    9. David L
    10. Fedorov LM
    11. Azuma Y
    12. Sears RC
    13. Dai MS

    (2021) The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis

    EMBO Reports 22:e50684.

    https://doi.org/10.15252/embr.202050684

    • PubMed
    • Google Scholar
76. 1. Scaiola A
    2. Peña C
    3. Weisser M
    4. Böhringer D
    5. Leibundgut M
    6. Klingauf-Nerurkar P
    7. Gerhardy S
    8. Panse VG
    9. Ban N

    (2018) Structure of a eukaryotic cytoplasmic pre-40S ribosomal subunit

    The EMBO Journal 37:e98499.

    https://doi.org/10.15252/embj.201798499

    • PubMed
    • Google Scholar
77. 1. Schäfer T
    2. Maco B
    3. Petfalski E
    4. Tollervey D
    5. Böttcher B
    6. Aebi U
    7. Hurt E

    (2006) Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit

    Nature 441:651–655.

    https://doi.org/10.1038/nature04840

    • PubMed
    • Google Scholar
78. 1. Scherl A
    2. Couté Y
    3. Déon C
    4. Callé A
    5. Kindbeiter K
    6. Sanchez JC
    7. Greco A
    8. Hochstrasser D
    9. Diaz JJ

    (2002) Functional proteomic analysis of human nucleolus

    Molecular Biology of the Cell 13:4100–4109.

    https://doi.org/10.1091/mbc.e02-05-0271

    • PubMed
    • Google Scholar
79. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
80. 1. Sloan KE
    2. Mattijssen S
    3. Lebaron S
    4. Tollervey D
    5. Pruijn GJ
    6. Watkins NJ

    (2013) Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing

    The Journal of Cell Biology 200:577–588.

    https://doi.org/10.1083/jcb.201207131

    • PubMed
    • Google Scholar
81. 1. Sloper-Mould KE
    2. Jemc JC
    3. Pickart CM
    4. Hicke L

    (2001) Distinct functional surface regions on ubiquitin

    The Journal of Biological Chemistry 276:30483–30489.

    https://doi.org/10.1074/jbc.M103248200

    • PubMed
    • Google Scholar
82. 1. Sowa ME
    2. Bennett EJ
    3. Gygi SP
    4. Harper JW

    (2009) Defining the human deubiquitinating enzyme interaction landscape

    Cell 138:389–403.

    https://doi.org/10.1016/j.cell.2009.04.042

    • PubMed
    • Google Scholar
83. 1. Strunk BS
    2. Novak MN
    3. Young CL
    4. Karbstein K

    (2012) A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

    Cell 150:111–121.

    https://doi.org/10.1016/j.cell.2012.04.044

    • PubMed
    • Google Scholar
84. 1. Sun XX
    2. He X
    3. Yin L
    4. Komada M
    5. Sears RC
    6. Dai MS

    (2015) The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc

    PNAS 112:3734–3739.

    https://doi.org/10.1073/pnas.1411713112

    • PubMed
    • Google Scholar
85. 1. Tafforeau L
    2. Zorbas C
    3. Langhendries JL
    4. Mullineux ST
    5. Stamatopoulou V
    6. Mullier R
    7. Wacheul L
    8. Lafontaine DL

    (2013) The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors

    Molecular Cell 51:539–551.

    https://doi.org/10.1016/j.molcel.2013.08.011

    • PubMed
    • Google Scholar
86. 1. Thevenon D
    2. Seffouh I
    3. Pillet C
    4. Crespo-Yanez X
    5. Fauvarque MO
    6. Taillebourg E

    (2020) A Nucleolar Isoform of the Drosophila Ubiquitin Specific Protease dUSP36 Regulates MYC-Dependent Cell Growth

    Frontiers in Cell and Developmental Biology 8:506.

    https://doi.org/10.3389/fcell.2020.00506

    • PubMed
    • Google Scholar
87. 1. Thomas F
    2. Kutay U

    (2003) Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway

    Journal of Cell Science 116:2409–2419.

    https://doi.org/10.1242/jcs.00464

    • PubMed
    • Google Scholar
88. 1. Trotta CR
    2. Lund E
    3. Kahan L
    4. Johnson AW
    5. Dahlberg JE

    (2003) Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

    The EMBO Journal 22:2841–2851.

    https://doi.org/10.1093/emboj/cdg249

    • PubMed
    • Google Scholar
89. 1. Turowski TW
    2. Lebaron S
    3. Zhang E
    4. Peil L
    5. Dudnakova T
    6. Petfalski E
    7. Granneman S
    8. Rappsilber J
    9. Tollervey D

    (2014) Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

    Nucleic Acids Research 42:12189–12199.

    https://doi.org/10.1093/nar/gku878

    • PubMed
    • Google Scholar
90. 1. Urbé S
    2. Liu H
    3. Hayes SD
    4. Heride C
    5. Rigden DJ
    6. Clague MJ

    (2012) Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions

    Molecular Biology of the Cell 23:1095–1103.

    https://doi.org/10.1091/mbc.E11-08-0668

    • PubMed
    • Google Scholar
91. 1. van der Veen AG
    2. Ploegh HL

    (2012) Ubiquitin-like proteins

    Annual Review of Biochemistry 81:323–357.

    https://doi.org/10.1146/annurev-biochem-093010-153308

    • PubMed
    • Google Scholar
92. 1. van Riggelen J
    2. Yetil A
    3. Felsher DW

    (2010) MYC as a regulator of ribosome biogenesis and protein synthesis

    Nature Reviews. Cancer 10:301–309.

    https://doi.org/10.1038/nrc2819

    • PubMed
    • Google Scholar
93. 1. Wada H
    2. Kito K
    3. Caskey LS
    4. Yeh ET
    5. Kamitani T

    (1998) Cleavage of the C-terminus of NEDD8 by UCH-L3

    Biochemical and Biophysical Research Communications 251:688–692.

    https://doi.org/10.1006/bbrc.1998.9532

    • PubMed
    • Google Scholar
94. 1. Widmann B
    2. Wandrey F
    3. Badertscher L
    4. Wyler E
    5. Pfannstiel J
    6. Zemp I
    7. Kutay U

    (2012) The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits

    Molecular Biology of the Cell 23:22–35.

    https://doi.org/10.1091/mbc.E11-07-0639

    • PubMed
    • Google Scholar
95. 1. Wild T
    2. Horvath P
    3. Wyler E
    4. Widmann B
    5. Badertscher L
    6. Zemp I
    7. Kozak K
    8. Csucs G
    9. Lund E
    10. Kutay U

    (2010) A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export

    PLOS Biology 8:e1000522.

    https://doi.org/10.1371/journal.pbio.1000522

    • PubMed
    • Google Scholar
96. 1. Woolford JL
    2. Baserga SJ

    (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae

    Genetics 195:643–681.

    https://doi.org/10.1534/genetics.113.153197

    • PubMed
    • Google Scholar
97. 1. Wyler E
    2. Zimmermann M
    3. Widmann B
    4. Gstaiger M
    5. Pfannstiel J
    6. Kutay U
    7. Zemp I

    (2011) Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies

    RNA 17:189–200.

    https://doi.org/10.1261/rna.2325911

    • PubMed
    • Google Scholar
98. 1. Wyler E
    2. Wandrey F
    3. Badertscher L
    4. Montellese C
    5. Alper D
    6. Kutay U

    (2014) The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14

    FEBS Letters 588:3685–3691.

    https://doi.org/10.1016/j.febslet.2014.08.013

    • PubMed
    • Google Scholar
99. 1. Ye Y
    2. Akutsu M
    3. Reyes-Turcu F
    4. Enchev RI
    5. Wilkinson KD
    6. Komander D

    (2011) Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21

    EMBO Reports 12:350–357.

    https://doi.org/10.1038/embor.2011.17

    • PubMed
    • Google Scholar
100. 1. Zemp I
     2. Wild T
     3. O'Donohue MF
     4. Wandrey F
     5. Widmann B
     6. Gleizes PE
     7. Kutay U

     (2009) Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2

     The Journal of Cell Biology 185:1167–1180.

     https://doi.org/10.1083/jcb.200904048

     • PubMed
     • Google Scholar
101. 1. Zemp I
     2. Wandrey F
     3. Rao S
     4. Ashiono C
     5. Wyler E
     6. Montellese C
     7. Kutay U

     (2014) CK1δ and CK1ε are components of human 40S subunit precursors required for cytoplasmic 40S maturation

     Journal of Cell Science 127:1242–1253.

     https://doi.org/10.1242/jcs.138719

     • PubMed
     • Google Scholar
102. 1. Zisser G
     2. Ohmayer U
     3. Mauerhofer C
     4. Mitterer V
     5. Klein I
     6. Rechberger GN
     7. Wolinski H
     8. Prattes M
     9. Pertschy B
     10. Milkereit P
     11. Bergler H

     (2018) Viewing pre-60S maturation at a minute's timescale

     Nucleic Acids Research 46:3140–3151.

     https://doi.org/10.1093/nar/gkx1293

     • PubMed
     • Google Scholar

Author details

1. Jasmin van den Heuvel

   1. Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland
   2. Molecular Life Sciences Ph.D. Program, Zurich, Switzerland

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Caroline Ashiono

   Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Ludovic C Gillet

   Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland

   Present address

   Institute of Molecular Systems Biology, Department of Biology, ETH Zurich, Zurich, Switzerland

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1001-3265

4. Kerstin Dörner

   1. Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland
   2. Molecular Life Sciences Ph.D. Program, Zurich, Switzerland

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

5. Emanuel Wyler

   Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland

   Present address

   Max‐Delbrück‐Center for Molecular Medicine in the Helmholtz Association (MDC), Institute for Medical Systems Biology (BIMSB), Berlin, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9884-1806

6. Ivo Zemp

   Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Supervision, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1549-9871

7. Ulrike Kutay

   Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   ulrike.kutay@bc.biol.ethz.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8257-7465

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