OTUD6 deubiquitination of RPS7/eS7 on the free 40 S ribosome regulates global protein translation and stress

Abstract

Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles.

Fig. 1. OTUD6 promotes resistance to alkylation and oxidation stress.

a Drosophila OTUD6 mutations and tagged endogenous forms. b OTUD6 catalytically inactive and loss-of-function mutants are sensitive to exposure to 0.05% MMS, measured at 32 h. Each data point represents the percent survival in a vial of 15 flies. One-way ANOVA/Dunnett’s, compared to control. (left: n = 14, 15, 15. right: n = 16, 16, 15, 15, 15, 16). c OTUD6 catalytically inactive and loss-of-function mutants are sensitive to exposure to 10 mM paraquat, measured at 72 h. One-way ANOVA/Dunnett’s, compared to control. (n = 16, 12, 12, 12, 16) d. Survival of third instar larvae exposed to X-ray irradiation. (n = 3, 3, 3). e Pavlovian short-term memory of aversive shock - neutral odor pairing. One-way ANOVA/Dunnett’s, compared to control. (n = 18, 18, 18). F OTUD6 is uniformly distributed in the Drosophila brain. Left: OTUD6.FLAG.HA detected with anti-HA (green). Right: untagged wild-type control. Scale bar: 50 μm. G Distribution of endogenously tagged OTUD6 (left) and OTUD6^C183A (right) in ovary egg chambers. Large nuclei and surrounding cytoplasm in the center are nurse cells that are surrounded by smaller follicle cells. Scale bar: 25 μm. Data are presented as mean values +/− SEM. Dots on on bar graphs and n represent biological replicates. Source data and statistics are provided as a Source Data file.

Fig. 2. OTUD6 physically Interacts with the 40 S ribosome and opposes 40 S ribosome ubiquitination.

a Volcano plot of OTUD6^C183A physical interactors, n = 3 biological replicates per genotype. b FLAG co-immunoprecipitation with tagged OTUD6^C183A followed by western analysis for RACK1 and OTUD6. c Genetic interaction analysis of heterozygous mutations for OTUD6 and Rack1. One way ANOVA with Sidak’s multiple comparisons test. (n = 24, 24, 20, 20, 24, 24) d E2 conjugating enzymes and E3 ligases tested for genetic interactions with OTUD6 for MMS sensitivity. e–g Suppressors of OTUD6^C183A MMS sensitivity. One way ANOVA with Sidak’s multiple comparisons test. (e: n = 15, 15, 14, 14. f: n = 20, 20, 20, 19, 13, 15. g: n = 20, 16, 17, 9). Data are presented as mean values +/− SEM. Dots on on bar graphs represent biological replicates. Source data and statistics are provided as a Source Data file.

Fig. 3. OTUD6 deubiquitinase activity promotes protein translation.

a Puromycin incorporation assay using Drosophila head lysate. Puromycin signal was normalized to tubulin. One-way ANOVA/Dunnett’s, compared to control. (n = 7, 6, 3). b OTUD6 mutants have extended lifespan. Representative graph of three independent experiments. Curves compared using log-rank (Mantel-Cox) test to control, OTUD6^C183A (P < 0.0001) and OTUD6^EP95 (P < 0.0001). (n = 75, 74, 45). c Developmental time, from egg to adulthood, is extended in OTUD6 mutants. Graph displays cumulative of eclosion to adult. One-way ANOVA/Dunnett’s, compared to control at day 10. (n = 8, 6, 6). d OTUD6 mutants have no impact on survival to adulthood. Data are presented as mean values +/− SEM. (n = 15, 13, 13). Dots on on bar graphs represent biological replicates. Source data and statistics are provided as a Source Data file.

Fig. 4. OTUD6 deubiquitinates monoubiquitinated RPS7.

a Serial monoubiquitin capture scheme. b Mass spectrometry analysis to identify differentially enriched proteins in monoubiquitin-selective capture reagents following a polyubiquitin preclear, OTUD6^C183A compared to genetic background control. Red indicates significant hits. n = 3 biological replicates per genotype. See Methods for statistical analysis. c Catalytically inactive OTUD6 mutants are enriched for RPS7 monoubiquitination. Serial monoubiquitin capture followed by western for RPS7 and total ubiquitin. Blots are representative of 3 biological replicates. d Lysate western for RPS7 (same as c), indicating monoubiquitination and no polyubiquitination. e Protein sequence of RPS7 in Drosophila melanogaster, Homo sapiens, and eS7A in Saccharomyces cerevisiae, showing the monoubiquitination site in yeast and the two residues that were mutated to arginine, K83R and K84R, to create Drosophila RPS7^K2R. f RPS7 monoubiquitination is absent in RPS7^K2R and OTUD6^C183A,RPS7^K2R flies. Blots are representative of 3 biological replicates. Data are presented as mean values +/− SEM. Source data and statistics are provided as a Source Data file.

Fig. 5. OTUD6 bidirectionally regulates the level of protein translation and RPS7 deubiquitination.

a Pan neuronal (elav-Gal4) RNAi knockdown (UAS-OTUD6.IR) and overexpression (UAS-OTUD6.OE) of OTUD6 resulted in decreased and increased puromycin incorporation, respectively, in fly heads. One-way ANOVA/Dunnett’s, compared to control. (n = 5,5,4). b Pan neuronal (elav-Gal4) RNAi knockdown (UAS-OTUD6.IR) and overexpression (UAS-OTUD6.OE) of OTUD6 resulted in increased and decreased RPS7 monoubiquitination in fly heads. Ubiquitinated RPS7 normalized to total ubiquitin. L: long exposure; S: short exposure. One-way ANOVA/Dunnett’s, compared to control. (n = 4, 4, 4). Data are presented as mean values +/− SEM. Dots on on bar graphs represent biological replicates. Source data and statistics are provided as a Source Data file.

Fig. 6. RPS7 monoubiquitination is regulated by OTUD6 interacting proteins and by MMS.

a RPS7 monoubiquitination in head extracts from OTUD6^C183A alone and in combination with mutations in Rack1, RNF10, and Cnot4. ANOVA/Dunnett’s, compared to OTUD6^C183A. (n = 4, 4, 3, 3). b RPS7 monoubiquitination is decreased by MMS treatment in head extracts. Two-tailed t-test compared to untreated controls. (n = 3, 3, 3, 3). c Levels of GFP, RPS7, and OTUD6.FLAG.HA in flies ubiquitously expressing GFP comprised of all common (GFP0D) and approximately 50% nonoptimal (GFP54C3’) codon content. One way ANOVA with Sidak’s multiple comparisons test. (Left: n = 8, 7, 6, 6. Middle: n = 6, 6. Right: n = 7, 7). d MMS sensitivity of OTUD6^C183A,RPS7^K2R flies. ANOVA/Sidak’s, planned comparisons. (n = 12, 12, 12, 12). e Protein translation levels detected by puromycin incorporation in OTUD6^C183A,RPS7^K2R flies. ANOVA/Dunnett’s, compared to control. (n = 7, 7, 7, 7) L: long exposure; S: short exposure. Data are presented as mean values +/− SEM. Dots on on bar graphs represent biological replicates. Source data and statistics are provided as a Source Data file.

Fig. 7. OTUD6 associates specifically with the RPS7-monoubiquitinated free 40 S ribosomal subunit to regulate 40 S ribosome occupancy on mRNA.

a Co-IP in the presence of cycloheximide with tagged OTUD6 and OTUD6^C183A, or untagged OTUD6^C183A as a control, probed for RPS7 (40 S), RPL11 (80 S), and eIF2α (43 S and 48 S preinitiation complexes). Blots are representative of 3 biological replicates. b Sucrose density fractionation and western detection of tagged OTUD6^C183A, RPS7, and RPL11. Blots are representative of 4 biological replicates. c Co-IP in the presence of cycloheximide with GFP-tagged RPL11 (UAS-Rpl11.GFP) expressed ubiquitously (Act5C-Gal4) in OTUD6^C183A.FLAG.HA flies, probed with HA, RPS7 and GFP. Blots are representative of 3 biological replicates. d Co-IP in the presence of cycloheximide with endogenously FLAG-tagged RPL3 in control and OTUD6^C183R, probed for RPS7, RPL11, and FLAG. Quantification of RPS7 non-ubiquitinated and monoubiquitinated forms. Mann-Whitney test (two-tailed). (n = 4, 4, 4, 4). e mRNA capture with oligo(dT) beads, probed by western analysis with antibodies for RPS7, RPS6, and RPL11. Ratio of OTUD6^C183A to genetic background control, except for RPS7 monoubiquitination. Mann-Whitney test (two-tailed). (n = 7, 7, 7, 7, 6, 6, 7, 7). L: long exposure; S: short exposure. Data are presented as mean values +/− SEM. Dots on on bar graphs represent biological replicates. Source data and statistics are provided as a Source Data file.

Fig. 8. OTUD6 protein levels are regulated by conditions that alter protein translation levels and efficacy.

a Western blot of OTUD6.FLAG.HA whole fly lysate probed for OTUD6 following 24 h 0.05% MMS exposure. Two-tailed t-test. (n = 8, 8). b OTUD6.FLAG.HA levels in young (3-5 days post eclosion) and old (30-35 days post eclosion) fly heads. Mann-Whitney test (two-tailed). (n = 5, 5). c Expression of poly(GR) repeat proteins in the Drosophila eye using GMR-Gal4, in OTUD6^C183A/+, with coexpression of OTUD6 RNAi (UAS-OTUD6.IR) or OTUD6 overexpression (UAS-OTUD6.OE). Example eye phenotypes. d Quantification of the poly(GR) eye phenotypes. One way ANOVA with Sidak’s multiple comparisons test. (n = 7, 21, 23, 13, 4). e Levels of OTUD6.FLAG.HA and RPS7 in head lysate of flies expressing poly(GR). OTUD6.FLAG.HA levels: Two-tailed t-test, RPS7 monoubiquitination: Mann-Whitney. L: long exposure; S: short exposure. Data are presented as mean values +/− SEM. (n = 4, 4, 4, 4). Dots on bar graphs represent biological replicates. Source data and statistics are provided as a Source Data file.