Polyubiquitin architecture editing on collided ribosomes maintains persistent RQC activity

Abstract

In ribosome-associated quality control (RQC), K63-linked polyubiquitination of ribosomal protein uS10 on the stalled ribosome is crucial for recruiting the RQC-trigger (RQT) complex. However, the mechanisms governing the maintenance and recycling of polyubiquitin architecture on colliding ribosomes remain unclear. Here we demonstrate that two deubiquitinating enzymes (DUBs), Ubp2 and Ubp3, play key roles in editing and recycling polyubiquitin chains on yeast uS10, thereby contributing to the promotion of RQC activity. Specifically, Ubp2 eliminates K63-linked polyubiquitin chains from uS10 on the free 40S subunit for recycling, while Ubp3 predominantly cleaves K48-linked di-ubiquitin and K48/K63-mixed-linkage polyubiquitin chains from uS10 on the translating ribosomes. We further demonstrate that K48-linkage-containing ubiquitin chains on uS10 of the colliding ribosome act as a negative signal for the RQT-mediated ribosome dissociation process. Collectively, our findings provide insight into the ubiquitin code in RQC, and define positive functions of two DUBs in maintaining persistent RQC activity.

Keywords: Deubiquitinase; Quality Control; Ribosome; Ubiquitin Code; Ubiquitination.

Figure 1. Deubiquitinating enzymes promote the RQC activity.

(A) The ubiquitination of uS10 increases in the ubp2∆, ubp3∆, and ubp2∆ubp3∆ mutants at the steady state. The ubiquitin level of uS10-3HA derived from plasmid pST001 in the wild-type (WT), ubp2∆, ubp3∆, and ubp2∆ubp3∆ mutants was detected with immunoblotting using an anti-HA antibody. The pixel intensity was measured by the plot profile tool of ImageJ. The intensity of the ubiquitinated uS10-3HA was normalized to the intensity of the eEF3 in each lane. Bar graphs represent the mean ± standard error (s.e.m.). Each dot represents an individual data point. Significance was calculated by Student’s t tests (n = 4; n represents the number of biological replicates). (B) The arrest products derived from GFP-R(CGN)12-FLAG-HIS3 reporter reduced in deubiquitinating enzymes deletion mutant cells. The arrest products in the indicated cells: wild-type, ltn1∆, ubp2∆, ubp2∆ltn1∆, ubp3∆, ubp3∆ltn1∆, ubp2∆ubp3∆, and ubp2∆ubp3∆ltn1∆, were detected by immunoblotting using an anti-GFP antibody (top panel). The pixel intensity was measured by the plot profile tool of ImageJ. The pixel intensity was measured by the plot profile tool of ImageJ. The intensity of the arrest products was normalized to the intensity of the eEF2 in each lane. Bar graphs represent the mean ± standard error (s.e.m.). Each dot represents an individual data point. Significance was calculated by Student’s t tests (n = 3; n represents the number of biological replicates). (C) Spot assay of the indicated cells in the presence of 10 μg/ml anisomycin. The indicating cells: wild-type, hel2∆, ubp2∆, ubp3∆, ubp2∆ubp3∆, diluted to OD₆₀₀ = 0.3, and tenfold serial dilutions were spotted and incubated at 30 °C for 2 days. (D) A collision sensor Hel2 is required for the arrest products in the deubiquitinating enzyme mutant cells. The arrest products in the indicated cells: wild-type, ltn1∆, ubp2∆, ubp2∆ltn1∆, ubp3∆, ubp3∆ltn1∆, hel2∆, hel2∆ltn1∆, hel2∆ubp2∆, hel2∆ubp2∆ltn1∆, hel2∆ubp3∆, and hel2∆ubp3∆ltn1∆, were detected by immunoblotting using an anti-GFP antibody (top panel). Total proteins used for immunoblottings in this Figure were prepared by TCA precipitation method. Source data are available online for this figure.

Figure 2. Substrate specificity of the deubiquitinating enzymes Ubp2 and Ubp3.

(A) The levels of the ubiquitinated uS10 in the deubiquitinating enzyme mutant cells. The total lysates prepared from wild-type, ubp2∆, ubp3∆, ubp2∆ubp3∆ expressing uS10-3HA from plasmid pST001, and subjected to polysome analysis. The levels of the ubiquitinated uS10-3HA in the fractions were detected by immunoblotting using an anti-HA antibody. (B) Quantification of ubiquitinated uS10 in (A). The pixel intensity was measured by the plot profile tool of ImageJ. The intensity of the ubiquitinated uS10-3HA was normalized to the intensity of the non-ubiquitinated uS10-3HA in each lane. (C) Statistical quantification analysis of ubiquitinated uS10 in the different ribosome fractions. The pixel intensity was measured by the plot profile tool of ImageJ. The intensity of the ubiquitinated uS10-3HA was normalized to the intensity of the non-ubiquitinated uS10-3HA in each lane. Bar graphs represent the mean ± standard error (s.e.m.). Each dot represents an individual data point. Significance was calculated by Student’s t tests (n = 3; n represents the number of biological replicates). (D) The non-K63-linked polyubiquitinated uS10 significantly increased in the ubp3∆ mutant cells but not in the ubp2∆ mutant cells. The ubiquitinated uS10-3HA in the ubi1∆ubi2∆ubi3∆ubi4∆ mutant (referred to as ubi1-4∆), as well as in the ubi1-4∆ubp2∆ and ubi1-4∆ubp3∆ mutant cells, was analyzed. These cells expressed either wild-type ubiquitin (Ub-WT) or the Ub-K63R mutant ubiquitin from plasmids pUB100 and pUB100-K63R, respectively. Detection was performed by immunoblotting using an anti-HA antibody. (E) In vitro deubiquitinating assay using 40S or polysome fractions of Ub-WT ubp2∆ strain or Ub-K63R ubp2∆ strains. The 40S or polysome fractions were prepared via sucrose gradient centrifugation. In the ubi1-4∆ubp2∆ mutant cells, Ub-WT or Ub-K63R was expressed from plasmids pUB100 or pUB100-K63R, respectively, while uS10-3HA was expressed from plasmid pST001. The purified Ubp2 or Ubp2C745S (catalytic mutant) in the indicated concentration was incubated with the fractions. After the incubation at 30 °C for 60 min, the uS10-3HA was detected by immunoblotting using an anti-HA antibody. (F) In vitro deubiquitinating assay using 40S or polysome fractions of Ub-WT ubp3∆ strain or Ub-K63R ubp3∆. The 40S or polysome fractions were prepared by sucrose gradient centrifugation. In the ubi1-4∆ubp3∆ mutant cells, Ub-WT or Ub-K63R was expressed from plasmids pUB100 or pUB100-K63R, respectively, whereas uS10-3HA was expressed from plasmid pST001. The purified Ubp3 or Ubp3C496A (catalytic mutant) in the indicated concentration was incubated with the fractions. After the incubation at 30 °C for 60 min, the polyubiquitinated uS10-3HA was detected by immunoblotting using an anti-HA antibody. Source data are available online for this figure.

Figure 3. Both K63- and K48-linked polyubiquitin chains on uS10 are increased in the ubp2 and ubp3 mutants.

(A) Schematic drawing of the uS10-3HA-His₆ purification. The uS10-3HA-His₆ was purified by a two-step affinity purification method. (B) Absolute quantification analysis of Ub chains of uS10. The uS10-3HA-His₆, uL23-FLAG, and Hel2-V5 were expressed from plasmids pST051, pKI191, and pST069, respectively, in the following cells: uS10∆, uS10∆ubp2∆, or uS10∆ubp3∆. The uS10-3HA-His₆ were purified by two-step purification and analyzed by a mass spectrometer. The abundance of Ub linkages was quantified using PRM. The data show the peptide abundance (fmol) of each Ub linkage signature peptide calculated by spiking in 25 fmol heavy isotope-labeled AQUA peptides (n = 3; n represents the number of biological replicates). (C) The ratio of the linkage types in (B). (D) Both K48- and K63-linked polyubiquitin chain was increased in the ubp2∆ or ubp3∆ mutant cells. The uS10-3HA-His₆, uL23-FLAG, and Hel2-V5 were expressed from plasmids pST051, pKI191, and pST069, respectively, in the following cells: uS10∆, uS10∆ubp2∆, or uS10∆ubp3∆. The uS10-3HA-His₆ were purified by two-step purification and subjected to immunoblotting using an anti-HA, K48-linkage-specific, and K63-linkage-specific anti-ubiquitin antibodies. (E) Schematic drawing of trypsin digestion for ubiquitin. Boxed peptide fragments were detected by Mass analysis. (F) Absolute quantification analysis of Ub chains of uS10 purified from the Ub-R54A mutants. The ubiquitinated uS10-3HA purified via a two-step purification method in the ubi1-4∆, ubi1-4∆ubp2∆ and ubi1-4∆ubp3∆ mutant cells, was analyzed by mass spectrometer. These cells expressed uS10-3HA-His₆, uL23-FLAG, Hel2-V5, and Ub-R54A mutant ubiquitin from plasmids pST081, pST097, pST109, and pUB100-R54A. The abundance of Ub linkages was quantified using PRM. The data show the peptide abundance (fmol) of each Ub linkage signature peptide calculated by spiking in 25 fmol heavy isotope-labeled AQUA peptides (n = 3; n represents the number of biological replicates). Source data are available online for this figure.

Figure 4. uS10 is ubiquitinated via K48- or K63-linked chains proximally, and via K63-linked chains distally.

(A) The affinity-purified ribosomes containing uS10-3HA-His₆ from the ubp2∆ uS10∆ cells were incubated with AMSH* (K63-linkage-specific deubiquitinase) and OTUB1* (K48-linkage-specific deubiquitinase). Immunoblotting was performed using an anti-HA antibody. (B) The affinity-purified ribosomes with uS10-3HA-His₆ from the ubp2∆uS10∆ mutant cells expressing uS10-3HA-His_6, uL23-FLAG, and Hel2-V5 from plasmids pST051, pKI191, and pST069, respectively, cells were incubated with AMSH*. Immunoblotting was performed using an anti-HA antibody (left), K48-linkage-specific anti-ubiquitin antibody (middle), and K63-linkage-specific anti-ubiquitin antibody (right). (C) Schematic drawing of ubiquitin chain linkage on uS10 and how Ubp2 trim ubiquitin chains. (D) The affinity-purified ribosomes containing uS10-3HA-His₆ from the ubp3∆uS10∆ mutant cells expressing uS10-3HA-His_6, uL23-FLAG, and Hel2-V5 from plasmids pST051, pKI191, and pST069, respectively, were incubated with AMSH* and OTUB1*. Immunoblotting was performed using an anti-HA antibody (Left panel). The ratio between mono- and di-ubiquitin of uS10 were plotted (Right panel). The pixel intensity was measured by the plot profile tool of ImageJ. Boxplots represent the distribution of values for each lane. The central line in each box indicates the median (50th percentile). The lower and upper bounds of the box correspond to the first (25th percentile) and third quartiles (75th percentile), respectively. The whiskers extend to the minimum and maximum values within 1.5 times the interquartile range (IQR) from the lower and upper quartiles. Individual data points, including potential outliers beyond this range, are overlaid as open circles with jitter for visualization. The intensity of the di-ubiquitinated uS10-3HA was normalized to the intensity of the mono-ubiquitinated uS10-3HA in each lane. Significance was calculated by Student’s t tests (n = 8; n represents the number of technical replicates). (E) The affinity-purified ribosomes containing uS10-3HA-His₆ from ubp3∆uS10∆ mutant cells expressing uS10-3HA-His_6, uL23-FLAG, and Hel2-V5 from plasmids pST051, pKI191, and pST069, respectively, were incubated with AMSH* and OTUB1*. Immunoblotting of uS10-3HA-His₆ performed using an anti-HA antibody (left), K48-linkage-specific anti-ubiquitin antibody (middle), and K63-linkage-specific anti-ubiquitin antibody (right). (F) Both K48- and K63-linked polyubiquitin chain was increased in the ubp2∆, ubp3∆, and ubp2∆ubp3∆ mutant cells. The uS10-3HA-His₆ purified via two-step purification from the following mutant cells: uS10∆, uS10∆ubp2∆, uS10∆ubp3∆, or uS10∆ubp2∆ubp3∆, expressing uS10-3HA-His_6, uL23-FLAG, and Hel2-V5 from plasmids pST051, pKI191, and pST069, respectively, were subjected to immunoblotting using an anti-HA, K48-linkage-specific, and K63-linkage-specific anti-ubiquitin antibodies. Source data are available online for this figure.

Figure 5. Investigation of the effects of proximal linkage type on the RQC activity using ubiquitin-fused uS10 (Ub-uS10) system.

(A) Schematic drawing of the Ub-uS10-3HA-His₆ construct. To construct Ub-uS10, the ubiquitin with G76V mutation was fused to 8–121 residues of uS10 that lack two lysine residues for Hel2-mediated ubiquitination. Two tag sequences, HA and six histidine residues (His₆) were inserted into C-terminus. (B) RQC is intact in cells expressing Ub-uS10 constructs. The arrest products derived from GFP-R(CGN)12-FLAG-HIS3 reporter in the uS10∆ltn1∆ cells expressing Ub-uS10 constructs (A) from plasmids pST158, pST159, pST160, pST161, pST162, pST163, pST164, or pST165 were detected by immunoblotting using an anti-GFP antibody (top panel). (C) Spot assay of uS10∆ cells expressing Ub-uS10 constructs (A) from plasmids pST158, pST159, pST160, pST161, pST162, pST163, pST164, or pST165 in the absence or presence of 10 μg/ml anisomycin. Cells diluted to OD₆₀₀= 0.3 and tenfold serial dilutions were spotted and incubated at 30°C for two days. (D) The analysis of the ubiquitination of Ub-uS10 constructs (A) expressing from plasmids pST158, pST159, pST160, pST161, pST162, pST163, pST164, or pST165 in the uS10∆ cell. Protein samples were prepared from the indicated cell expressing Ub-uS10 wild-type or the mutations. The polyubiquitinated uS10 was detected with immunoblotting using an anti-HA antibody. (E) Hel2 is responsible for the K63-linked ubiquitination of Ub-uS10. The analysis of the ubiquitination of Ub-uS10 constructs (A) expressing from plasmids pST158, pST159, pST160, pST161, pST162, pST163, pST164, or pST165 in the uS10∆ or uS10∆hel2∆ cell. The ubiquitinated uS10 was detected as in (D). (F–H) Ufd4 is solely responsible for the formation of the K29-linked polyubiquitin chain. (F) The analysis of the ubiquitination of Ub-uS10 constructs (A) expressing from plasmids pST158, pST159, pST160, pST161, pST162, pST163, pST164, or pST165 in the uS10∆ or uS10∆ufd4∆ cell. The ubiquitinated uS10 was detected as in (D). (G, H) Absolute quantification analysis of Ub chains of uS10 purified from the indicated mutants. The abundance of Ub linkages was quantified using PRM. The data show the peptide abundance (fmol) of each Ub linkage signature peptide calculated by spiking in 25 fmol heavy isotope-labeled AQUA peptides (n = 3; n represents the number of biological replicates). (I) Ufd4 does not serve in RQC. The arrest products derived from GFP-R(CGN)12-FLAG-HIS3 reporter in the ufd4∆ or ufd4∆ltn1∆ cells were detected by immunoblotting using an anti-GFP antibody (top panel). Total proteins used for all immunoblotting in this Figure were prepared by TCA precipitation method (B, D, E, F, I). Source data are available online for this figure.

Figure 6. The in vitro reconstitution of the K48/K63-mixed ubiquitin chain-conjugated colliding ribosomes.

(A, C, E) In vitro reconstitution assay for colliding ribosomes: The purified RNCs from the in vitro translation (IVT) reaction of the His-SDD1 model mRNA using the IVT extract prepared from Ub-K63only-uS10 mutant strain (A): uS10∆ski2∆ expressing Ub-K63only-uS10-3HA from plasmid pST320, Ub-K48only-uS10 mutant strain (C): uS10∆ski2∆ expressing Ub-K48only-uS10-3HA from plasmid pST321, or Ub-K48only-uS10 expressing hel2∆ mutant strain (E): uS10∆ski2∆hel2∆ expressing Ub-K48only-3HA from plasmid pST321, were separated by sucrose density gradient centrifugation and detected by UV absorbance at a wavelength of 260 nm. HA-tagged uS10 in each fraction was detected by immunoblotting using an anti-HA antibody. (B, D) Ub-CRest assay: The purified RNCs from the in vitro translation (IVT) of His-SDD1 model mRNA using the IVT extract prepared from Ub-K63only-uS10 mutant strain (B) or Ub-K48only-uS10 mutant strain (D), were incubated with AMSH*. Immunoblotting was performed using an anti-HA antibody. (F) Pull-down assay of the trimer RQT complex with the Ub4 (K63-K63-K63), Ub4 (K48-K48-K48), and the Ub4 (K63-K48-K63): The RQT complex was immobilized on IgG magnetic beads and mixed with each Ub4. After the binding and washing step, the proteins in the final elution were separated by 10% Nu-PAGE and detected by anti-ubiquitin and anti-FLAG antibodies. Source data are available online for this figure.

Figure 7. RQT complex dissociates the collided ribosome with the K63-linked polyubiquitin chain but not K48-K63-mixed polyubiquitin chain on Ub-uS10.

(A, B) In vitro splitting assay using different linkage types conjugated colliding ribosomes: The K48/K63-mixed linkage or the K63-linked polyubiquitin chain-conjugated RNCs were purified IVT reaction of His-SDD1 model mRNA using the extract prepared from Ub-K48only-uS10 mutant strain (A): uS10∆ski2∆ expressing Ub-K48only-uS10-3HA from plasmid pST321, or Ub-K63only-uS10 mutant strain (B) uS10∆ski2∆ expressing Ub-K63only-uS10-3HA from plasmid pST320. Subsequently, the purified RNCs were mixed with or without the RQT complex in the presence of ATP and incubated for 45 min at 25 °C. After the reaction, RNCs were separated by sucrose density gradient centrifugation. HA-tagged uS10 in each fraction was detected by immunoblotting using an anti-HA antibody. Source data are available online for this figure.

Figure EV1. Ubp2 and Ubp3 are involved in the deubiquitination of uS10.

(A) Spot assay of the indicated uS10 tagged or untagged strains. The indicating cells: uS10∆ expressing uS10, uS10-3HA, uS10-3HA-His₆, or Ub-uS10-3HA-His₆ from plasmids pKI124, pKI236, pST051, and pST158, respectively, diluted to OD₆₀₀ = 0.3 and 10-fold serial dilutions were spotted and incubated at 30°C for two days. (B) Genetic screening to identify the deubiquitinating enzymes for uS10. Protein samples prepared from the indicated mutant cells: wild-type, ubp1∆, ubp2∆, ubp3∆, ubp4∆, ubp5∆, ubp6∆, ubp7∆, ubp8∆, ubp9∆, ubp11∆, ubp12∆, ubp13∆, ubp14∆, ubp15∆, ubp16∆, otu1∆, otu2∆, and yuh1∆, expressing uS10-3HA from plasmid pST001 were subjected to immunoblotting using an anti-HA antibody. Total proteins used for the immunoblotting were prepared by Cell lysis method. (C) The analysis of the ubiquitination of the uS10-K6/8 R mutant in the ubp2∆ and ubp3∆ strains. The ubiquitin level of uS10-3HA or uS10K6/8R-3HA derived from plasmid pKI236 or pKI237 in the uS10∆, uS10∆ubp2∆, uS10∆ubp3∆ mutants was detected with immunoblotting using an anti-HA antibody.

Figure EV2. Ubp2 and Ubp3 are associated with 40S subunit and polysome, respectively.

(A, B) The total lysates derived from 3 x HA genomic tagging Ubp2 (A) or Ubp3 (B) expressing cells were subjected to the sucrose density gradient and sedimented through by ultracentrifugation. For Ubp3, lysates were prepared either with or without micrococcal nuclease (MNase) treatment. Ubp2-3HA and Ubp3-3HA in each fraction were detected by immunoblotting using an anti-HA antibody. (C) The polyubiquitination of uS10 in K6R and K8R mutant of uS10. The 3 x HA-tagged uS10, uS10-K6/8 R, uS10-K6R or K8R was expressed from plasmids pKI237, pKI238, and pKI239, respectively, in the uS10∆ cells and detected the HA-tagged uS10 by immunoblotting using an anti-HA antibody. Total proteins used for immunoblotting were prepared by Cell lysis method. (D) CBB stain of purified Ubp2, Ubp2 C745S, Ubp3, Ubp3C496A.

Figure EV3. The polyubiquitin chains formed on uS10 are mainly K63- and K48-linkage.

(A) The two-step purification of uS10-3HA-His₆. The uS10-3HA-His_6, uL23-FLAG, and Hel2-V5 were expressed from plasmids pST051, pKI191, and pST069 in uS10∆ cells. The ubiquitinated uS10-3HA-His₆ were purified by two-step affinity purification. The purified samples were stained with CBB (Left) or subjected to Immunoblotting using an anti-HA antibody, anti-V5, and anti-FLAG antibody (Right). (B) The sample preparation for Absolute quantification analysis of Ub chains. The uS10-3HA-His₆ or uS10-K6/8R-3HA-His₆ together with uL23-FLAG, and Hel2-V5 were expressed from plasmids pST051, pST052, pKI191, and pST069 in the following strains: uS10∆, uS10∆ubp2∆, uS10∆ubp3∆. The ubiquitinated uS10-3HA-His₆ were purified by two-step affinity purification. The purified samples were stained with CBB (Left) or subjected to Immunoblotting using an anti-HA antibody (Right). (C) Schematic drawing of K48/K63 mixed or branched ubiquitin chain architectures. (D) The sample preparation for Absolute quantification analysis of Ub chains. The uS10-3HA-His₆ or uS10-K6/8R-3HA-His₆ together with uL23-FLAG, Hel2-V5, and Ub-R54A were expressed from plasmids pST051, pST052, pKI191, pST069, and pUB100-R54A in the following strains: ubi1-4∆uS10∆, ubi1-4∆uS10∆ubp2∆, ubi1-4∆uS10∆ubp3∆. The ubiquitinated uS10-3HA-His₆ were purified by two-step affinity purification. The purified samples were subjected to Immunoblotting using an anti-HA antibody.

Figure EV4. The K48- and K63-linked di-ubiquitin chains are formed on uS10-K6R.

(A) K48-linked or K63-linked tetraubiquitin chains were reacted with AMSH* (K63-linkage-specific deubiquitinase) and OTUB1* (K48-linkage specific deubiquitinase). The protein samples were separated by 15% Nu-PAGE and detected by silver staining (top panel) or immunoblotting using an anti-ubiquitin antibody (bottom panel). (B) Both K48- and K63-linked polyubiquitin chain was formed on uS10. The uS10-WT-3HA-His₆ or uS10-K6R-3HA-His₆ together with uL23-FLAG and Hel2-V5 were expressed from plasmids pST051, pST137, pKI191, and pST069, respectively, in uS10∆ cells. The uS10-WT-3HA-His₆ or uS10-K6R-3HA-His₆ were purified by two-step affinity purification. Immunoblotting of purified samples using an anti-HA antibody, K48-linkage-specific anti-ubiquitin antibody, and K63-linkage-specific anti-ubiquitin antibody. (C) Schematic drawing of the architecture of K48/K63 mixed ubiquitin chain via K48-linked di-ubiquitination.

Figure EV5. The sample preparation for Absolute quantification analysis of Ub chains in the ufd4∆ mutant.

The uS10-3HA-His₆, uL23-FLAG, and Hel2-V5 were expressed from plasmids pST051, pKI191, and pST069 in the following strains: uS10∆, uS10∆ufd4∆, uS10∆ubp2∆, uS10∆ufd4∆. The ubiquitinated uS10-3HA-His₆ were purified by two-step affinity purification. The purified samples were subjected to Immunoblotting using an anti-HA antibody.

Figure EV6. RQT complex is associated with the K63-linked ubiquitinated colliding ribosomes but not with K48-K63-mixed polyubiquitinated colliding ribosomes.

The purified RNCs from the in vitro translation (IVT) reaction of the His-SDD1 model mRNA using the IVT extract prepared from Ub-K48only-uS10 mutant strain (A): uS10∆ski2∆ expressing Ub-K48only-uS10-3HA from plasmid pST321, or Ub-K63only-uS10 mutant strain (B): uS10∆ski2∆ expressing Ub-K63only-uS10-3HA from plasmid pST320, were incubated with the RQT complex in the absence of ATP and then separated by sucrose density gradient centrifugation. The ribosome abundance was detected by UV absorbance at 260 nm. HA-tagged uS10 and Flag-tagged Slh1, the component of the RQT complex, in each fraction, were detected by immunoblotting using anti-HA and anti-Flag antibodies, respectively.