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. 2017 Jul 31;8(1):159.
doi: 10.1038/s41467-017-00188-1.

Ubiquitination of stalled ribosome triggers ribosome-associated quality control

Yoshitaka Matsuo  1 , Ken Ikeuchi  1 , Yasushi Saeki  2 , Shintaro Iwasaki  3   4 , Christian Schmidt  5 , Tsuyoshi Udagawa  1 , Fumiya Sato  1 , Hikaru Tsuchiya  2 , Thomas Becker  5 , Keiji Tanaka  2 , Nicholas T Ingolia  3 , Roland Beckmann  5 , Toshifumi Inada  6
Affiliations

Ubiquitination of stalled ribosome triggers ribosome-associated quality control

Yoshitaka Matsuo et al. Nat Commun. .

Abstract

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Hel2-dependent ubiquitination was required for a triggering step of RQC pathway. a An E3 ubiquitin ligase Hel2 was required for not only translation arrest, but also the production of arrest products. b The ubiquitination activity of Rqt1 was crucial for induction of RQC. c The distribution of Rqt1 proteins in polysome profiles. d The ubiquitinated proteins in ribosome depended on Hel2/Rqt1 and Ubc4. Arrowed proteins were identified by mass spectrometry. e K212 residue of uS3 and K6 and K8 residues of uS10 were ubiquitination sites of them. f Hel2-dependent ubiquitination of uS10-K6/8 was crucial for induction of RQC pathway
Fig. 2
Fig. 2
RQC requires three novel ribosome-quality control trigger factors (RQT). a Identification of RQT factors. Purified Rqt1-ribosome complex were analyzed by SDS-PAGE. Arrowed proteins were identified by mass spectrometry. b RQT factors played a crucial role in triggering step of RQC pathway. c Rqt2, Rqt3, and Rqt4 were dispensable for the ubiquitination of ribosome proteins by Rqt1. d Affinity purification of Rqt2-, Rqt3-, and Rqt4-ribosome complexes. e Rqt2, Rqt3, and Rqt4 formed a complex independent of ribosomes
Fig. 3
Fig. 3
The mutations of Rqt3 in the residues crucial for the ubiquitin-binding domain diminished RQC. a Alignment of the conserved residues in the CUE domains of Rqt3, Vps9, and Cue2. The mutated residues in Rqt3-Ub-m were shown in red. b CUE domain of Rqt3 interacted directly with ubiquitin, and it was abolished by Ub-m mutations. c The mutations of Rqt3 in the residues crucial for the ubiquitin-binding domain diminished RQC. d The mutation in the ubiquitin binding of Rqt3 is dispensable for the association of Rqt2-4 in Rqt1-ribosome complex. e The distribution of Rqt2-4 proteins in polysome profiles of Rqt3-Ub-m mutant. f The expression levels of endogenously HA-tagged Rqt2 and Rqt4, Flag-tagged Rqt3 and Rqt3-Ub-m proteins in Rqt3-Ub-m mutant cells
Fig. 4
Fig. 4
The mutations of Rqt2 in the conserved residues in ATPase domain diminished RQC. a Alignment of the conserved residues in the RecA1 motif I of Slh1/Rqt2, Brr2, Ski2, and hRad51. The substitution of the lysine to arginine residue of motif I in human Rad51 diminished the ATPase activity. b The mutations of Rqt2 in the conserved residues in ATPase domain diminished RQC. c Rqt2-3 were distributed in heavy polysome fraction in Rqt2K316R mutant. d The expression levels of endogenously HA-tagged Rqt2, Rqt2-K316R, Rqt3, and Rqt4 proteins in Rqt2-K316R mutant cells
Fig. 5
Fig. 5
Stalled ribosomes in a rotated state conformation are targets for RQC. a The anisomycin sensitivity of rqt and uS10-K6/8R mutants. b Ribosome short footprints were accumulated at CGA-CGA codons. The short footprints and long footprints from the ribosome profiling data set were mapped on the reporter gene (GFP-R12-FLAG-HIS3). The reads were plotted at approximate position of ribosome A-site. c Translation of HIS3 downstream of R(CGN)12 arrest-sequence was increased in the rqt and uS10-K6/8R mutants. d Codon specificity of poly-arginine sequence to induce translation arrest and RQC. e Specificity of di-codons for accumulation of short and long footprints. f GFP-X-FLAG-HIS3 reporter assay of Top 10 di-codons in the accumulation of short footprints. g GFP-X-FLAG-HIS3 reporter assay of top 4 di-codons in the accumulation of long footprints
Fig. 6
Fig. 6
Rqt1-bound ribosomes are the rotated form with hybrid tRNAs. a CBB-stained SDS gel and Amidoblack-stained blot of pullouts from Rqt1-FTP strains. b Particle sorting was done by 3D classification in RELION using four classes starting from 142,001 particles. This resulted in three dominant classes with rotated ribosomes (two classes in rotated state 1 and one class in rotated state 2) and one class with un-rotated (classical) state ribosomes; the highest-resolution close-to-focus data set was refined using movie processing (see “Methods” section). In total, the data set contained 22.4% classical state and 77.6% rotated state ribosomes. c Population of programmed and unprogrammed ribosomes in Rqt1-FTP and uL30-TAP pullouts
Fig. 7
Fig. 7
ASCC3, a human homolog of yeast Rqt2 is required for RQC induced by poly(A) sequence. a Rqt1 factors were required for RQC induced by poly-lysine sequences in yeast. b The AAA repeat induces RQC in mammalian cells. c Schematic drawing of alignment of the domain structure of ZNF598/hRqt1 and ASCC3/hRqt2. d ZNF598 and ASCC3 were localized mainly in cytosol. e ZNF598 was co-purified with ASCC3. f ZNF598 is involved in translation repression and the production of RQC substrate by K(AAA)24 sequence. g ASCC3 is a homolog of Rqt2 in mammalian cells
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