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. 2021 Feb 5;24(3):102145. doi: 10.1016/j.isci.2021.102145

The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation

Yuka Takehara 1,5, Hideki Yashiroda 1,5, Yoshitaka Matsuo 2, Xian Zhao 1, Akane Kamigaki 1, Tetsuo Matsuzaki 1,6, Hidetaka Kosako 3, Toshifumi Inada 2,4, Shigeo Murata 1,7,∗
PMCID: PMC7900223  PMID: 33665564

Summary

Ubiquitination is a major post-translational modification of ribosomal proteins. The role of ubiquitination in the regulation of ribosome functions is still being elucidated. However, the importance of ribosome deubiquitination remains unclear. Here, we show that the cycle of ubiquitination and deubiquitination of the 40S ribosome subunit eS7 is important for efficient translation. eS7 ubiquitination at lysine 83 is required for efficient protein translation. We identified Otu2 and Ubp3 as the deubiquitinating enzymes for eS7. An otu2Δubp3Δ mutation caused a defect in protein synthesis. Ubp3 inhibited polyubiquitination of eS7 in polysomes to keep eS7 in a mono-ubiquitinated form, whereas Otu2 was specifically bound to the free 40S ribosome and promoted the dissociation of mRNAs from 40S ribosomes in the recycling step. Our results provide clues for understanding the molecular mechanism of the translation system via a ubiquitination-deubiquitination cycle.

Subject areas: biological sciences, biochemistry, molecular biology

Graphical abstract

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Highlights

  • •

    eS7 ubiquitination at lysine 83 is required for efficient protein translation

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    Ubp3 prevents eS7 from polyubiquitination in 80S ribosomes and polysomes

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    Otu2 deubiquitinates mono-ubiquitinated eS7 in the 40S ribosome

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    Otu2 facilitates dissociation of mRNAs from 40S ribosomes in ribosome recycling

Biological sciences; biochemistry; molecular biology

Introduction

The ribosome is a crucial component in the process of translation in all living cells. The eukaryotic 80S ribosome comprises two subunits, the large 60S ribosomal subunit and the small 40S ribosomal subunit. These two subunits cyclically assemble and disassemble in the translation process. The translation process comprises four phases: initiation, elongation, termination, and recycling (Dever et al., 2016; Jackson et al., 2010). In the initiation phase, initiation factors (eIF1, eIF1A, eIF2, eIF3, and eIF5) and Met-tRNAiMet are recruited to the 40S ribosome to form a 43S preinitiation complex (43S PIC). Next, the 43S PIC attaches to the capped 5′-end of mRNA with the cap-binding complex eIF4F, forming a 48S preinitiation complex (48S PIC) (Eliseev et al., 2018). Then, the joining of the 60S ribosome to the 48S PIC is mediated by eIF5B to produce an 80S initiation complex that is competent for protein synthesis (Hinnebusch, 2014). During the elongation phase, translating 80S ribosomes form polysomes on a single molecule of mRNA. In the termination phase, eukaryotic release factor 1 (eRF1) recognizes the stop codon and releases the synthesized polypeptide chain (Hellen, 2018). In the recycling phase, the 80S post-termination complex (80S PTC) is dissociated into a free 40S and 60S ribosomes, deacylated tRNA, and mRNA to initiate new rounds of translation by a two-step process of recycling. In the first step of recycling, the ATPase Rli1 (yeast)/ABCE1 (mammals) forces a split of the 80S PTC into a free 60S ribosome and a tRNA/mRNA-bound 40S ribosome (Becker et al., 2012; Young and Guydosh, 2019; Young et al., 2015). In the second step of recycling, tRNA and mRNA are released from the 40S ribosome to produce a free 40S ribosome.

Recent studies suggest that ubiquitination and deubiquitination of the ribosome play crucial roles in controlling translation. For example, ubiquitination of uS10 is required for ribosome-associated quality control (RQC), leading to the dissociation of 80S ribosomes into 40S and 60S ribosomes (Ikeuchi et al., 2019; Matsuo et al., 2017). Sequential ubiquitination of uS3 is required for 18S nonfunctional ribosomal RNA decay (Sugiyama et al., 2019). Ubiquitinated 40S ribosomal proteins are deubiquitinated to escape from lysosomal degradation (Meyer et al., 2020). Deubiquitination of uL25 is required for the 60S ribosome ribophagy upon nutrient starvation (Ossareh-Nazari et al., 2014). Even during constitutive translation, many ribosomal proteins are reportedly ubiquitinated and most likely deubiquitinated (Mayor et al., 2005, 2007; Peng et al., 2003; Tagwerker et al., 2006). However, the biological significance and mechanisms of ubiquitination and especially deubiquitination during constitutive translation remain largely unknown.

Ubiquitination is carried out by three types of enzymes: El ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases (Amerik and Hochstrasser, 2004; Haq and Ramakrishna, 2017). Of these enzymes, E3 ligases are responsible for substrate recognition and mediate transfer of ubiquitin onto the lysine residues of targeted proteins. Ubiquitination can be reversed by deubiquitinating enzymes (DUBs). DUBs cleave off ubiquitin from ubiquitin-conjugated proteins. Budding yeast DUBs are classified into four superfamilies based on the architecture of their catalytic domains. Three of them, ubiquitin-specific protease (USP) family, ubiquitin C-terminal hydrolase (UCH) family, and ovarian tumor protease (OTU) family, belong to the cysteine protease family. The remaining Jab1/MPN/Mov34 (JAMM) family is a metalloprotease family. Although the functions of many E3 ligases are known, DUB functions remain elusive.

Otu2 is a potential DUB of the OTU family that was identified as a 40S ribosome-associated protein by a global mass spectrometry (Fleischer et al., 2006). However, the role of Otu2 in ribosome function remains unknown. In this study, we identified Otu2 and Ubp3 as deubiquitinating enzymes for eS7 and propose that the cycle of ubiquitination and deubiquitination of eS7 is important for efficient protein synthesis.

Results

Otu2 is associated with 40S ribosomes

Otu2 was previously identified as a 40S ribosome-associated protein. To confirm the association of Otu2 with 40S ribosomes, we prepared cells endogenously expressing C-terminally 3×Flag-tagged Otu2 (Otu2-3×Flag). The cell extract was fractionated using sucrose density gradient (SDG) centrifugation. Immunoblot analysis using anti-Flag antibodies showed that Otu2 was distributed specifically in the 40S ribosome fractions, consistent with the previous report (Figure 1A).

Figure 1.

Figure 1

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Otu2 is associated with 40S ribosomes

(A) Total cell extract from the cells endogenously expressing C-terminally 3×Flag-tagged Otu2 (Otu2-3×Flag) was fractionated using 10%–40% sucrose density gradient (SDG) centrifugation. Absorbance at 254 nm of each fraction was measured to quantify the contents of ribosomes. The distribution patterns of Otu2-3×Flag were investigated by immunoblotting with anti-Flag antibodies.

(B) Proteins immunoprecipitated with anti-Flag M2 beads were subjected to SDS-PAGE followed by silver staining.

(C) Western blot analysis of immunoprecipitates with Otu2-3×Flag by using anti-GFP antibodies (top panel) and anti-Flag antibodies (lower panel).

We next analyzed Otu2-3×Flag immunoprecipitates using mass spectrometry. Multiple 40S ribosomal proteins were identified as Otu2-binding proteins as expected. Of note, we identified Rli1, a ribosome-associated factor, which was the most prominent binding protein other than 40S ribosomal proteins (Figure 1B). Rli1 recycles terminating ribosomes and controls translation initiation. The interaction of Rli1 with Otu2 was further confirmed by immunoblot analysis using strains endogenously expressing GFP-tagged Rli1 and Otu2-3×Flag (Figure 1C). These results suggest that Otu2 is associated with 40S ribosomes at least, in the recycling and initiation steps of the translation cycle, in which Rli1 plays a role.

Otu2 and Ubp3 have overlapping functions in efficient protein synthesis

Single deletion of Otu2 did not cause apparent growth defect under normal culture conditions (Figure 2A). Deubiquitinating enzymes often have overlapping functions, which mask the effect of each single deletion. Thus, we crossed the otu2Δ strain with all other 18 single DUB deletion strains and found that otu2Δ caused a growth defect when combined with ubp3Δ (Figure 2A), indicating that Otu2 has overlapping function with Ubp3.

Figure 2.

Figure 2

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Otu2 and Ubp3 have overlapping functions in efficient protein synthesis

(A) Each strain was streaked on a YPAD plate and incubated for 2 days at 28°C. See also Figure S1.

(B) Total cell extracts were fractionated using 10%–40% SDG centrifugation. Absorbance at 254 nm of each fraction was measured to quantify the contents of ribosomes.

(C) Efficiency of the protein synthesis in each strain was monitored by measuring the ability to incorporate L-homopropargylglycine (HPG) into nascent proteins (n = 3, mean ± SD). HPG is an amino acid analog of methionine containing an alkyne moiety. Cycloheximide (CHX) was used as a translation inhibitor.

The deubiquitinating activity of the Ubp3 has been shown previously (Baker et al., 1992), but whether Otu2 is a bona fide DUB has remained unexplored. Exploiting the otu2Δubp3Δ growth defect, we examined whether the conserved cysteine 178 in the OTU domain of Otu2 was essential for the Otu2 function. Whereas wild-type (WT) Otu2 rescued the otu2Δubp3Δ growth defect, expression of catalytically inactive Otu2 mutant (Otu2-C178S) did not (Figure S1A). Furthermore, overexpression of Otu2-C178S in the ubp3Δ strain caused growth defect (Figure S1B). These results suggest that Otu2 acts as a genuine deubiquitinating enzyme and that Otu2 and Ubp3 have a common substrate for deubiquitination. We also noticed that overexpression of Otu2-C178S had a deleterious effect even in otu2Δubp3Δ cells, which indicates that there are overlapping deubiquitinating enzymes other than Ubp3 (Figure S1C).

Ubp3 is also thought to interact with the ribosome, especially with 60S ribosomes and polysomes (Fleischer et al., 2006). We speculated that Otu2 and Ubp3 have overlapping functions related to the ribosome and compared the ribosome profiles of WT, otu2Δ, ubp3Δ, and otu2Δubp3Δ cells by SDG analysis. Although both ribosome patterns of single otu2Δ and ubp3Δ mutant cells were similar to that of WT cells, otu2Δubp3Δ cells decreased the amount of polysomes, suggesting that protein synthesis is defective in otu2Δubp3Δ cells (Figure 2B).

To confirm that Otu2 and Ubp3 are involved in the synthesis of nascent proteins, the incorporation of L-homopropargylglycine, an amino acid analog of methionine containing an alkyne moiety, was monitored (Mittal et al., 2017). Compared with the strain complemented with both the OTU2 and UBP3 genes, the efficiency of protein synthesis was decreased by approximately 50% by the lack of UBP3, although no apparent decrease was observed by the lack of OTU2 alone (Figure 2C). The defect in protein synthesis was prominent in otu2Δubp3Δ cells, in which a 90% decrease in the efficiency was observed (Figure 2C). These results indicate that cooperative deubiquitination by Otu2 and Ubp3 is required for efficient protein synthesis.

Otu2 and Ubp3 deubiquitinate eS7A in the distinct ribosome complexes

To identify potential substrates for Otu2, the plasmid encoding Flag-6×His-tagged ubiquitin was expressed in WT, otu2Δ, ubp3Δ, and otu2Δubp3Δ strains, and the cell extracts were fractionated by SDG centrifugation. Immunoblotting analysis using anti-Flag antibodies revealed that the ubiquitinated proteins of ~34 kDa that were observed in the 80S and polysome fractions in the WT strain emerged in the 40S ribosome fractions of the otu2Δ strain (Figure 3A). Further accumulation of these proteins in the 40S fractions was observed in the otu2Δubp3Δ strain. These results suggest that the ~34 kDa ubiquitinated proteins are ribosome-associated proteins that may be deubiquitinated by Otu2 and Ubp3.

Figure 3.

Figure 3

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Otu2 and Ubp3 deubiquitinate eS7A in the distinct ribosome complexes

(A) Distribution patterns of ubiquitinated proteins. Total cell extracts from Flag-6×His-Ub-expressing cells were fractionated using 10%–40% SDG centrifugation. Each fraction was subjected to immunoblot analysis with anti-Flag antibodies.

(B) Ubiquitinated proteins purified with Ni-NTA beads from 40S ribosome fractions were subject to SDS-PAGE followed by the immunoblot analysis using anti-Flag antibodies (left panel) and Coomassie Brilliant Blue (CBB) staining (right panel). See also Figure S2.

(C) Immunoblot analysis of eS7A-HA with anti-HA (upper panel) and anti-ubiquitin (lower panel) antibodies. The asterisk indicates another potential substrate of Otu2 and Ubp3.

(D) Distribution patterns of eS7A-HA. Total cell extracts were fractionated using 10%–40% SDG centrifugation. Each fraction was subjected to immunoblot analysis with anti-HA antibodies.

We then purified ubiquitinated proteins from 40S ribosome fractions of each strain. The ~34 kDa ubiquitinated proteins were purified most efficiently from the 40S fractions of otu2Δubp3Δ cells and not from those of WT and ubp3Δ cells (Figure 3B). Subsequent mass spectrometry analysis revealed that eS7A and eS7B were included in the corresponding bands (Figure S2A). eS7A and eS7B are 87% identical in amino acid sequence and are both 40S ribosomal proteins. To examine whether Otu2 and Ubp3 were involved in deubiquitination of eS7, hemagglutinin (HA)-tagged eS7A was expressed in the eS7AΔ and the otu2Δubp3ΔeS7AΔ strains. eS7A was detected as a major 28 kDa band, the predicted molecular weight for eS7A, and a minor 34 kDa band by anti-HA antibodies in eS7AΔ cells (Figure 3C). On the other hand, the 34 kDa band became more apparent in otu2Δubp3ΔeS7AΔ cells. This 34 kDa band was also detected using anti-ubiquitin antibodies in otu2Δubp3ΔeS7AΔ cells (Figure 3C). The 34 kDa band also accumulated through the overexpression of Otu2-C178S in the ubp3Δ strain (Figure S2B). These results indicate that Otu2 and Ubp3 are involved in deubiquitination of eS7.

To determine the ubiquitination sites of eS7A and eS7B, we analyzed the 34 kDa ubiquitinated proteins in the ubp3Δ strain expressing Otu2-C178S by mass spectrometry. We identified lysines 56 and 83 as potential ubiquitination sites of eS7A and eS7B (Figures S2B–S2D). We then substituted lysines 56 and 83 with arginines in eS7A and expressed the resultant eS7A-K56R and -K83R in the eS7AΔ and the otu2Δubp3ΔeS7AΔ strains. Mono- and possibly di-ubiquitinated forms of eS7A were eliminated by the K83R mutation in both strains, whereas the K56R mutation did not affect the ubiquitination status of eS7A (Figure 3C). These results indicate that K83 is the major ubiquitination site of eS7A and that Otu2 and Ubp3 deubiquitinate K83-ubiquitinated eS7.

To verify that the 34 kDa ubiquitinated proteins that accumulate in the 40S fractions in cells lacking Otu2 were ubiquitinated eS7, cell lysates fractionated by SDG centrifugation were blotted for eS7A-HA. In WT cells, mono-ubiquitinated eS7A was observed in the 80S and the polysome fractions, as reported previously (Figure 3D). In otu2Δ and Otu2-C178S-expressing cells, mono-ubiquitinated eS7A emerged in the 40S ribosome fraction in addition to the 80S and the polysome fractions. Single deletion of Ubp3 caused accumulation of di-ubiquitinated eS7A in the 80S and the polysome fractions (Figure 3D). These results suggest that Otu2 and Ubp3 deubiquitinate eS7 in the distinct ribosome complexes; Otu2 deubiquitinates mono-ubiquitinated eS7 in 40S ribosomes to the non-ubiquitinated form, whereas Ubp3 deubiquitinates eS7 in 80S ribosomes and polysomes to the mono-ubiquitinated form.

Meanwhile, simultaneous loss of Otu2 and Ubp3 activities resulted in the accumulation of di-ubiquitinated eS7A in the 40S ribosome fraction. This is consistent with the idea that these entities are dissociated products of 80S ribosomes in the absence of Ubp3 and that Otu2 can deubiquitinate di-ubiquitinated eS7 in free 40S ribosomes. However, we cannot exclude the possibility that eS7 is ubiquitinated in nascent 40S ribosomes, and ubiquitinated eS7 in nascent 40S ribosomes was also accumulated in otu2Δ cells.

Deubiquitination of eS7 by Otu2 is required for efficient translation in vitro

To examine whether Otu2 deubiquitinates eS7A directly in the 40S ribosome, recombinant His-Otu2 was added to mono- and di-ubiquitinated eS7A-3HA prepared from 40S ribosome fractions of otu2Δubp3ΔeS7A-3HA cells. When His-Otu2 was added at 6.25 nM or above, both mono- and di-ubiquitinated eS7A proteins disappeared. On the other hand, the addition of His-Otu2-C178S had no effect on ubiquitinated eS7A. GST-Ubp3 and its inactive form GST-Ubp3-C469A also did not change the ubiquitination status of eS7A in the 40S ribosomes (Figure 4A). These results suggest that Otu2, but not Ubp3, directly deubiquitinates eS7 in the 40S ribosome.

Figure 4.

Figure 4

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Deubiquitination of eS7 by Otu2 is required for efficient translation in vitro

(A) In vitro deubiquitination assay of eS7A-3HA using indicated recombinant deubiquitinating enzymes and 40S ribosome fractions prepared from otu2Δubp3Δski2ΔeS7A-3HA cells. The asterisk indicates a non-specific band.

(B) In vitro translation assay using recombinant His-Otu2 (WT and C178S), synthesized Renilla luciferase mRNAs, and a yeast in vitro translation extract obtained from the otu2Δubp3Δski2ΔeS7A-3HA strain (n = 4, mean ± SD, Student’s t test, ∗p < 0.05, n.s.: not significant). Ski2 was deleted to prevent Renilla luciferase mRNAs from degradation.

(C) Detection of the ubiquitination status of eS7A-3HA in the in vitro translation extract assayed in (B) by immunoblot analysis with anti-HA antibodies. See also Figure S3.

As shown in Figure 2C, protein synthesis was severely impaired in otu2Δubp3Δ cells. We then examined whether the defect in protein synthesis in otu2Δubp3Δ cells was relieved by recombinant His-Otu2. We added His-Otu2 and in vitro transcribed reporter mRNAs encoding Renilla luciferase to cell extracts prepared from the otu2Δubp3ΔeS7A-3HA strain and measured in vitro translation activity. Luciferase expression was clearly increased by the addition of His-Otu2 at 62.5 nM but not by the same concentration of His-Otu2-C178S (Figure 4B). We also confirmed that the increase in luciferase expression was accompanied with deubiquitination of eS7A (Figure 4C). These results suggest that deubiquitination of eS7 by Otu2 is directly required for efficient protein translation.

However, the precise mechanism by which recombinant Otu2 upregulates the synthesis of Renilla luciferase (Figure 4B) remains unknown. We speculate that Out2 prevents abortive translation by transiently associating with 80S ribosomes. To confirm this, we expressed β-galactosidase and GFP double-tagged with Flag at the N-terminus and V5 at the C-terminus to detect possible truncated translation products caused by abortive translation in WT and Δotu2 cells. However, immunoblot analysis results reveal no difference in band patterns after using anti-Flag and anti-V5 antibodies, even when cells were treated with the proteasome inhibitor bortezomib, which inhibits the degradation of unstable truncated translation products (Figure S3). These results suggest that the deletion of Otu2 does not cause abortive translation.

Ubiquitination of lysine 83 in eS7A is important for translation

Although we had revealed the importance of eS7 deubiquitination in 40S ribosomes for protein translation, the significance of eS7 ubiquitination that constantly existed in the translating 80S ribosomes and polysomes remained unknown. To investigate the biological importance of eS7 ubiquitination, the efficiency of protein synthesis was measured in the WT, eS7AΔ, and eS7A-K83R strains. The K83R mutation decreased the synthesis of nascent proteins to the same degree as the eS7AΔ mutation (Figure 5A). We further confirmed that the K83R mutation blocked the ubiquitination of eS7A (Figure 5B). This result indicates that ubiquitination of lysine 83 in eS7A in 80S ribosomes and polysomes during the translation process is important for translation.

Figure 5.

Figure 5

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Ubiquitination of lysine 83 in eS7A is important for translation

(A) The eS7AΔ strain was transformed with pRS315 (vector), p315-7A (pRS315-eS7A-HA), or pUT156 (pRS315-eS7A-K83R-HA). Efficiency of the protein synthesis in each transformant was monitored by measuring the ability to incorporate L-homopropargylglycine (HPG) into nascent proteins as in Figure 2C (n = 3, mean ± SD, Student’s t test, ∗∗p < 0.01, ∗p < 0.05). Cycloheximide (CHX) is used as a translation inhibitor.

(B) Detection of the ubiquitination status of eS7A-HA in each strain assayed in (A) by immunoblot analysis with anti-HA antibodies.

Otu2 binds to recycled 40S ribosomes and the 43S preinitiation complex but not to the 48S preinitiation complex

After dissociation of 80S ribosomes into 40S and 60S at the translation termination, 40S ribosomes exist from the recycling to translation initiation steps in the translation cycle (Figures 6A). It is possible that Otu2 plays a role during ribosome recycling because it was specifically distributed in the 40S ribosome fractions (Figure 1). During the process from recycling to translation initiation, various ribosome-associated factors bind to and dissociate from 40S ribosomes in order, forming 43S- and 48S-preinitiation complexes (PIC). To more precisely determine which status of 40S ribosomes Otu2 is associated with, we examined which of the binding proteins characteristic of each status of 40S ribosomes was coimmunoprecipitated with Otu2. Rli1 and Hcr1 are recycling factors associated with the 80S PTC, the post-termination and free 40S ribosomes, and the 43S preinitiation complex (43S PIC) (Beznosková et al., 2013; Young and Guydosh, 2019). eIF3A is a subunit of the eIF3 complex, which acts from ribosome splitting through translation initiation (Valasek et al., 2003). eIF5 and eIF2 with Met-tRNAiMet are recruited to the free 40S ribosome to form the 43S PIC. eIF4A and eIF4E are subunits of the cap-binding eIF4F complex in the 48S PIC (Figures 6A and 6B) (Dever et al., 2016). Of these proteins, Rli1, Hcr1, eIF3A, eIF5, eIF2α, and eIF2γ were coimmunoprecipitated with Otu2, whereas eIF4A and eIF4E were not (Figure 6C). These results suggest that Otu2 binds to post-termination and free 40S ribosomes in the recycling process and 43S PICs, but Otu2 is released from 43S PICs before 48S PICs are formed.

Figure 6.

Figure 6

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Otu2 binds to recycled 40S ribosomes and the 43S preinitiation complex

(A) Schematic model for the translation cycle in Saccharomyces cerevisiae. Only proteins and complexes, which appear in the main text, are depicted.

(B) Diagram shows the interactions between the ribosome and the ribosome-associated proteins examined in (C). The dotted lines of Hcr1 and Rli1 indicate the uncertainty of when Hcr1 and Rli1 are dissociated from the initiation complex.

(C) Immunoblot analysis of coimmunoprecipitated proteins with Flag-Otu2.

Otu2 plays a crucial role in dissociation of mRNAs from 40S ribosomes in ribosome recycling

Translated mRNAs are released from 40S ribosomes in the recycling process (Figure 6A). To further specify the status of Otu2-bound 40S ribosomes, we examined whether mRNAs were bound to 40S ribosomes purified with Otu2. 40S ribosomes were pulled down from 40S ribosome fractions of the strain expressing endogenously tagged uS15-3×Flag (Figure 7A). Similarly, Otu2-bound 40S ribosomes were purified from 40S ribosome fractions of the strain expressing the Otu2-3×Flag. Importantly, 48S PICs, which contain new activated mRNAs, should not be included in Otu2-bound 40S ribosomes based on the results in Figure 6C. Nevertheless, RT-qPCR analysis using RNAs extracted from each purified 40S ribosome revealed that mRNA levels of all the tested housekeeping genes (ACT1, PGK1, eS27A, and eS27B) were higher in 40S ribosomes purified with Otu2-3×Flag than those purified with uS15-3×Flag (Figure 7B). These results suggest that Otu2 mainly binds to 40S ribosomes before releasing mRNAs.

Figure 7.

Figure 7

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Otu2 plays a crucial role in dissociation of mRNAs from 40S ribosomes in ribosome recycling

(A) Experimental procedure for (B). Whole free and Otu2-bound 40S ribosomes were purified with anti-Flag antibodies from 40S ribosome fractions of the strains expressing uS15-3×Flag and Otu2-3×Flag, respectively.

(B) mRNA levels in the 40S ribosomes purified with uS15- and Otu2-3×Flag examined by RT-qPCR analysis (n = 3, mean ± SD, Student’s t test, ∗p < 0.05).

(C) Experimental procedure for (D).

(D) mRNA levels in the 40S and 80S ribosomes purified with uS15-3×Flag from WT and otu2Δ cells examined by RT-qPCR analysis (n = 3, mean ± SD, Student’s t test, ∗∗p < 0.01, ∗p < 0.05, n.s.: not significant). RDN18-1 was used as the endogenous reference gene for internal controls (B and D).

We then investigated whether Otu2 deficiency caused an increase in mRNA-bound 40S ribosomes. RNAs were extracted from 40S and 80S ribosomes purified from the WT and otu2Δ strains expressing uS15-3×Flag, and PGK1 and ACT1 mRNA levels were examined by RT-qPCR (Figure 7C). Although no or a slight increase in PGK1 and ACT1 mRNA levels was detected in the 80S ribosomes of otu2Δ cells, mRNA levels of both genes were much higher in 40S ribosomes from otu2Δ cells than in those from WT cells (Figure 7D). These results suggest that post-termination 40S ribosomes are accumulated by the deletion of Otu2, and deubiquitination of eS7A of post-termination 40S ribosomes by Otu2 plays a role in the dissociation of mRNAs from 40S ribosomes during ribosome recycling.

Discussion

Based on the present study, we propose a model in which ubiquitination and deubiquitination of eS7 promotes the translation cycle (Figure 6A). eS7 is ubiquitinated in the 80S ribosome for efficient translation (Figure 5), whereas Ubp3 deubiquitinates eS7 to keep it as a mono-ubiquitinated form (Figure 3). After translation termination, 80S ribosomes are split into 40S and 60S ribosomes. Otu2 forms a stable complex with the 40S ribosome and deubiquitinates mono-ubiquitinated eS7 into non-ubiquitinated eS7, which triggers dissociation of translated mRNAs and serves to reinitiate a second round of translation (Figures 3 and 7). Thus, the cycle of mono-ubiquitination and deubiquitination of eS7 in the 80S/polysome and the 40S ribosome, respectively, plays an important role for efficient translation.

It has been shown that Not4 is an E3 ligase responsible for ubiquitination of eS7A. Polysomes were decreased in the Not4-deleted strain, suggesting that eS7A ubiquitination is important for efficient translation (Panasenko and Collart, 2012). Panasenko and Collart (2012) also showed simultaneous mutations of five lysine residues (K72, K76, K83, K84, and K101) to arginine in eS7A-abrogated eS7A ubiquitination by Not4. However, it has remained uncertain which lysine residue was actually ubiquitinated and responsible for efficient translation. We identified K83 as the major ubiquitination site of eS7 by mass spectrometry and site-directed mutagenesis (Figures 3C and S2D). Translation was remarkably repressed in the strain expressing eS7A-K83R, suggesting that ubiquitination of eS7 at K83 is critical for the normal translation cycle (Figure 5). Recently, Buschauer et al. (2020) showed that eS7 ubiquitination by Not4 is involved in the function of Not5 in sensing codon optimality by using the eS7-4KR (K72, 76, 83, 84R) mutant. eS7 ubiquitination helps Not5 associate with ribosomes (Buschauer et al., 2020). Among the four lysine residues, K83 may be essential for this association. It has also been shown that eS7 could be ubiquitinated at K84 by Not4 and that eS7 ubiquitination at K84 is equivalent to that at K83 for RQC-uncoupled no-go decay (Ikeuchi et al., 2019). The reason we did not observe eS7 ubiquitination at K84 in the eS7A-K83R mutant may have been because of the different contexts or because the K83R mutation affected K48 ubiquitination. In addition, we observed translation repression in otu2Δubp3Δ cells, accompanied by accumulation of poly-ubiquitinated eS7 (Figures 2 and 3). This observation indicates that the deubiquitination of eS7 is also required for efficient translation. Although both Otu2 and Ubp3 deubiquitinate eS7, Otu2 and Ubp3 play distinct roles in different ribosome complexes.

We showed that Ubp3 maintains eS7 in a mono-ubiquitinated form and prevents eS7 from polyubiquitination in 80S ribosomes and polysomes. eS7 is reported to undergo polyubiquitination by the E3 ligase Hel2 when K6 and K8 of eS10 are mutated to arginine to suppress translation for triggering a quality control mechanism (Ikeuchi et al., 2019). It is considered that translation is repressed due to activation of the quality control mechanism when eS7 is polyubiquitinated. Thus, it is conceivable that Ubp3 has a function to prevent activation of an incorrect quality control mechanism by keeping the state of mono-ubiquitination of eS7 during translation.

Our findings suggest that Otu2 facilitates dissociation of mRNAs from 40S ribosomes through deubiquitination of eS7 in the ribosome recycling step for a new round of translation. mRNAs were enriched in Otu2-bound 40S ribosomes, compared with whole free 40S ribosomes, and deletion of Otu2 increased 40S ribosome-associated mRNAs (Figure 7). However, several important questions remain unanswered. One of these questions is how Otu2 binds specifically to the 40S ribosome. It is unlikely that this specific binding of Otu2 to the 40S ribosome is mediated by recognition of ubiquitinated eS7s, because ubiquitinated eS7s also exist in 80S ribosomes. eS7 interacts with eL19 in the 80S ribosome (Jenner et al., 2012; Klinge et al., 2012; Wilson and Doudna Cate, 2012). Thus, Otu2 may bind to eS7 at the same site as eL19. Structural analysis of the 40S ribosome with Otu2 will be helpful to address this issue. Another question is how deubiquitination of eS7 promotes dissociation of mRNAs from 40S ribosomes. The mechanism for dissociation of tRNA and mRNA from the 40S ribosome remains uncertain (Pisarev et al., 2010; Skabkin et al., 2010; Young et al., 2018). At present, Tma64/eIF2D, Tma20/MCT-1, and Tma22/DENR are most likely to play an important role in the release of mRNAs from 40S ribosomes. It has been reported that unrecycled 40S ribosomes reinitiate translation at AUGs in the 3′ UTR or re-associate with 60S ribosomes in an AUG-independent manner in the tma mutants (Young et al., 2018). These unusual events might have occurred in otu2Δ cells and may be one of the reasons why 40S ribosomes were not accumulated in the otu2Δ strain (Figure 2B). Thus, it is interesting to examine whether Otu2 and mono-ubiquitin on eS7 affects the affinity and activity of Tma proteins to recycling 40S ribosomes.

Although it has been well established that ubiquitination of various ribosomal proteins is required for many critical aspects in translational control (Dougherty et al., 2020), we have demonstrated for the first time that eS7 is cyclically ubiquitinated and deubiquitinated in the normal translation process. Our results reveal another layer of regulation for ribosome functions in which a ubiquitination-deubiquitination cycle plays a key role.

Limitations of the study

In this study, we demonstrated that deubiquitination of eS7 by Otu2 was involved in mRNA release from the 40S ribosome. However, it remains unclear how deubiquitination of eS7A mediates dissociation of mRNAs from 40S ribosomes. We also demonstrated that eS7 was mono-ubiquitinated in 80S ribosomes and polysomes and that mono-ubiquitination of eS7 at K83 is required for efficient translation. Ubp3 prevented eS7 from polyubiquitination. However, it remains unclear how Ubp3 leaves eS7 mono-ubiquitinated and why the mono-ubiquitination of eS7 is required for efficient translation. We should also consider the importance of mRNA co-translational decay as a regulatory mechanism for translation cycle in our future study.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Shigeo Murata (smurata@mol.f.u-tokyo.ac.jp).

Materials availability

All unique/stable reagents generated in this study are available from the Lead contact with a completed Materials Transfer Agreement.

Data and code availability

This study did not generate datasets.

Methods

All methods can be found in the accompanying Transparent methods supplemental file.

Acknowledgments

This work was supported by JSPS KAKENHI (Grant Numbers JP18H05500, JP18H04022 for S.M., JP18KK0229, JP19H04966 and JP20K06628 for H.K., JP18H03977, JP19H05281 for T.I., and JP19K06481 for Y.M.), AMED (Grant Number 19gm1110003 AMED-CREST for S.M.), and Joint Usage and Joint Research Programs, the Institute of Advanced Medical Sciences, Tokushima University.

Author contributions

Conceptualization, Y.T., H.Y., and S.M.; Methodology, Y.T., H.Y., Y.M., H.K., T. I., and S.M.; Investigation, Y.T., H.Y., Y.M., X.Z., A.K., T.M., and H.K.; Writing—Original Draft, Y.T., H.Y., and S.M.; Funding Acquisition, Y.M., H.K., T.I., and S.M.; Project Administration, S.M.; Resources, Y.T., H.Y., Y.M., and A.K.; Supervision, S.M.

Declaration of interests

The authors declare no competing interests.

Published: March 19, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102145.

Supplemental information

Document S1. Transparent methods, Figures S1–S3, and TablesS1–S4
mmc1.pdf (1.2MB, pdf)

References

  1. Amerik A.Y., Hochstrasser M. Mechanism and function of deubiquitinating enzymes. Biochim. Biophys. Acta. 2004;1695:189–207. doi: 10.1016/j.bbamcr.2004.10.003. [DOI] [PubMed] [Google Scholar]
  2. Baker R.T., Tobias J.W., Varshavsky A. Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family. J. Biol. Chem. 1992;267:23364–23375. [PubMed] [Google Scholar]
  3. Becker T., Franckenberg S., Wickles S., Shoemaker C.J., Anger A.M., Armache J.-P., Sieber H., Ungewickell C., Berninghausen O., Daberkow I. Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature. 2012;482:501–506. doi: 10.1038/nature10829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beznosková P., Cuchalová L., Wagner S., Shoemaker C.J., Gunišová S., von der Haar T., Valášek L.S. Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells. PLoS Genet. 2013;9:e1003962. doi: 10.1371/journal.pgen.1003962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buschauer R., Matsuo Y., Sugiyama T., Chen Y.H., Alhusaini N., Sweet T., Ikeuchi K., Cheng J., Matsuki Y., Nobuta R. The Ccr4-Not complex monitors the translating ribosome for codon optimality. Science. 2020;368:eaay6912. doi: 10.1126/science.aay6912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dever T.E., Kinzy T.G., Pavitt G.D. Mechanism and regulation of protein synthesis in Saccharomyces cerevisiae. Genetics. 2016;203:65–107. doi: 10.1534/genetics.115.186221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dougherty S.E., Maduka A.O., Inada T., Silva G.M. Expanding role of ubiquitin in translational control. Int. J. Mol. Sci. 2020;21:1151. doi: 10.3390/ijms21031151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eliseev B., Yeramala L., Leitner A., Karuppasamy M., Raimondeau E., Huard K., Alkalaeva E., Aebersold R., Schaffitzel C. Structure of a human cap-dependent 48S translation pre-initiation complex. Nucleic Acids Res. 2018;46:2678–2689. doi: 10.1093/nar/gky054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fleischer T.C., Weaver C.M., McAfee K.J., Jennings J.L., Link A.J. Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes. Genes Dev. 2006;20:1294–1307. doi: 10.1101/gad.1422006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haq S., Ramakrishna S. Deubiquitylation of deubiquitylases. Open Biol. 2017;7:170016. doi: 10.1098/rsob.170016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hellen C.U.T. Translation termination and ribosome recycling in eukaryotes. Cold Spring Harb. Perspect. Biol. 2018;10:a032656. doi: 10.1101/cshperspect.a032656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hinnebusch A.G. The scanning mechanism of eukaryotic translation initiation. Annu. Rev. Biochem. 2014;83:779–812. doi: 10.1146/annurev-biochem-060713-035802. [DOI] [PubMed] [Google Scholar]
  13. Ikeuchi K., Tesina P., Matsuo Y., Sugiyama T., Cheng J., Saeki Y., Tanaka K., Becker T., Beckmann R., Inada T. Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways. EMBO J. 2019;38:e100276. doi: 10.15252/embj.2018100276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jackson R.J., Hellen C.U.T., Pestova T.V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 2010;11:113–127. doi: 10.1038/nrm2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jenner L., Melnikov S., Garreau de Loubresse N., Ben-Shem A., Iskakova M., Urzhumtsev A., Meskauskas A., Dinman J., Yusupova G., Yusupov M. Crystal structure of the 80S yeast ribosome. Curr. Opin. Struct. Biol. 2012;22:759–767. doi: 10.1016/j.sbi.2012.07.013. [DOI] [PubMed] [Google Scholar]
  16. Klinge S., Voigts-Hoffmann F., Leibundgut M., Ban N. Atomic structures of the eukaryotic ribosome. Trends Biochem. Sci. 2012;37:189–198. doi: 10.1016/j.tibs.2012.02.007. [DOI] [PubMed] [Google Scholar]
  17. Matsuo Y., Ikeuchi K., Saeki Y., Iwasaki S., Schmidt C., Udagawa T., Sato F., Tsuchiya H., Becker T., Tanaka K. Ubiquitination of stalled ribosome triggers ribosome-associated quality control. Nat. Commun. 2017;8:159. doi: 10.1038/s41467-017-00188-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mayor T., Graumann J., Bryan J., MacCoss M.J., Deshaies R.J. Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol. Cell. Proteomics. 2007;6:1885–1895. doi: 10.1074/mcp.M700264-MCP200. [DOI] [PubMed] [Google Scholar]
  19. Mayor T., Lipford J.R., Graumann J., Smith G.T., Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol. Cell. Proteomics. 2005;4:741–751. doi: 10.1074/mcp.M400220-MCP200. [DOI] [PubMed] [Google Scholar]
  20. Meyer C., Garzia A., Morozov P., Molina H., Tuschl T. The G3BP1-family-USP10 Deubiquitinase complex rescues ubiquitinated 40S subunits of ribosomes stalled in translation from lysosomal degradation. Mol. Cell. 2020;77:1193–1205.e1195. doi: 10.1016/j.molcel.2019.12.024. [DOI] [PubMed] [Google Scholar]
  21. Mittal N., Guimaraes J.C., Gross T., Schmidt A., Vina-Vilaseca A., Nedialkova D.D., Aeschimann F., Leidel S.A., Spang A., Zavolan M. The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan. Nat. Commun. 2017;8:457. doi: 10.1038/s41467-017-00539-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ossareh-Nazari B., Niño C.A., Bengtson M.H., Lee J.-W., Joazeiro C.A.P., Dargemont C. Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy. J. Cell Biol. 2014;204:909–917. doi: 10.1083/jcb.201308139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Panasenko O.O., Collart M.A. Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase. Mol. Microbiol. 2012;83:640–653. doi: 10.1111/j.1365-2958.2011.07957.x. [DOI] [PubMed] [Google Scholar]
  24. Peng J., Schwartz D., Elias J.E., Thoreen C.C., Cheng D., Marsischky G., Roelofs J., Finley D., Gygi S.P. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 2003;21:921–926. doi: 10.1038/nbt849. [DOI] [PubMed] [Google Scholar]
  25. Pisarev A.V., Skabkin M.A., Pisareva V.P., Skabkina O.V., Rakotondrafara A.M., Hentze M.W., Hellen C.U.T., Pestova T.V. The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol. Cell. 2010;37:196–210. doi: 10.1016/j.molcel.2009.12.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Skabkin M.A., Skabkina O.V., Dhote V., Komar A.A., Hellen C.U.T., Pestova T.V. Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev. 2010;24:1787–1801. doi: 10.1101/gad.1957510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sugiyama T., Li S., Kato M., Ikeuchi K., Ichimura A., Matsuo Y., Inada T. Sequential ubiquitination of ribosomal protein uS3 triggers the degradation of non-functional 18S rRNA. Cell Rep. 2019;26:3400–3415.e3407. doi: 10.1016/j.celrep.2019.02.067. [DOI] [PubMed] [Google Scholar]
  28. Tagwerker C., Flick K., Cui M., Guerrero C., Dou Y., Auer B., Baldi P., Huang L., Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking. Mol. Cell. Proteomics. 2006;5:737–748. doi: 10.1074/mcp.M500368-MCP200. [DOI] [PubMed] [Google Scholar]
  29. Valasek L., Mathew A.A., Shin B.S., Nielsen K.H., Szamecz B., Hinnebusch A.G. The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo. Genes Dev. 2003;17:786–799. doi: 10.1101/gad.1065403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wilson D.N., Doudna Cate J.H. The structure and function of the eukaryotic ribosome. Cold Spring Harb. Perspect. Biol. 2012;4:a011536. doi: 10.1101/cshperspect.a011536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Young D.J., Guydosh N.R. Hcr1/eIF3j is a 60S ribosomal subunit recycling accessory factor in vivo. Cell Rep. 2019;28:39–50.e34. doi: 10.1016/j.celrep.2019.05.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Young D.J., Guydosh N.R., Zhang F., Hinnebusch A.G., Green R. Rli1/ABCE1 recycles terminating ribosomes and controls translation reinitiation in 3'UTRs in vivo. Cell. 2015;162:872–884. doi: 10.1016/j.cell.2015.07.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Young D.J., Makeeva D.S., Zhang F., Anisimova A.S., Stolboushkina E.A., Ghobakhlou F., Shatsky I.N., Dmitriev S.E., Hinnebusch A.G., Guydosh N.R. Tma64/eIF2D, tma20/MCT-1, and Tma22/DENR recycle post-termination 40S subunits in vivo. Mol. Cell. 2018;71:761–774 e765. doi: 10.1016/j.molcel.2018.07.028. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods, Figures S1–S3, and TablesS1–S4
mmc1.pdf (1.2MB, pdf)

Data Availability Statement

This study did not generate datasets.

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