Yeast strains

Yeast strains are listed in Supplementary Table S1. Yeast Saccharomyces cerevisiae W303-1a-based strains were obtained by established recombination techniques using PCR-amplified cassette sequences (kanMX4, hphMX4, natMX4, natNT2 or HISMX6) (12,15). To construct strains carrying essential ribosomal protein genes (uS10, uS3 and eS7AeS7B), the shuffle strain transformed with plasmids expressing mutant ribosomal protein products was grown on an SDC plate containing 0.5 mg/ml of 5-fluoro-orotic acid (5-FOA, #F9001-5, Zymo Research, Irvine, CA, USA), and URA3 absent strains were isolated.

Plasmids

Plasmids used in this study are listed in Supplementary Table S2. DNA cloning was performed by PCR amplification with gene-specific primers using PrimeSTAR HS DNA polymerase (#R010A, Takara-bio, Shiga, Japan) and T4 DNA ligase (#M0202S, NEB, Ipswich, MA, USA). All cloned DNAs amplified by PCR were verified by sequencing.

Yeast culture and media

Yeast cells were cultured with YPD or synthetic complete (SC) medium with 2% glucose at 30°C, and harvested at log phase by centrifugation after discarding the medium.Cell pellets were frozen in liquid nitrogen immediately after harvest and stored at –80°C until used.

RNA isolation for determination

Total RNA solutions were prepared using the acidic phenol RNA extraction method as follows: the cell pellet was resuspended with 300 μl RNA buffer (300 mM NaCl, 20 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS, dissolved in DEPC-treated MilliQ water at room temperature) on ice, followed by immediate addition of 300 μl water-saturated phenol and vortex mixing for 10 s. The mixture was incubated at 65°C for 5 min, mixed by vortex for 10 s and chilled on ice for 5 min. After centrifugation at 16 000 × g for 5 min at room temperature, 300 μl of the water layer was transferred to a new 1.5 ml RNase-free tube. After addition of 250 μl water-saturated phenol, the procedure was repeated; 300 μl of the water layer was transferred to a new 1.5 ml RNase-free tube, and 300 μl water-saturated phenol/chloroform (1:1) was added, followed by a 10 s vortex and centrifugation at 16 000 × g for 5 min at room temperature. Then, 300 μl of the water layer was transferred to a new 1.5 ml RNase-free tube, and 300 μl water-saturated phenol/chloroform/isoamylalchol (25:24:1) was added, followed by a 10 s vortex and centrifugation at 16 000 × g for 5 min at room temperature. Then, 300 μl of the water layer was transferred to a new 1.5 ml RNase-free tube and subjected to ethanol precipitation. The RNA pellet was finally dissolved with 20–30 μl DEPC-treated water to obtain a total RNA solution.

RNA electrophoresis and northern blotting

A volume of 6 μl (2.5 μg) total RNA solution was mixed with 24 μl glyoxal mix [600 μl DMSO, 200 μl deionised 40% glyoxal, 120 μl of 10 × MOPS buffer (200 mM MOPS, 50 mM NaOAc, 10 mM EDTA pH 7.0), 62.5 μl of 80% glycerol and 17.5 μl DEPC-treated water in 1 ml] and 3 μl RNA loading buffer (50% glycerol, 10 mM EDTA pH 8.0, 0.05% bromophenol blue and 0.05% xylene cyanol). The mixture was incubated at 74°C for 10 min, followed by incubation on ice for 10 min to obtain RNA samples. Aliquots of 25 μl of each sample were electrophoresed at 200 V for 40 min on a 1.2% or 2% agarose gel in 1× MOPS buffer (20 mM MOPS, 5 mM NaOAc, 1 mM EDTA pH 7.0), followed by transfer of RNA to Hybond-N+ membranes (GE healthcare, Chicago, IL, USA) with 20× SSC (3 M NaCl and 300 mM trisodium citrate dihydrate) for 18 h using a capillary system. RNA was cross-linked on the membrane using a CL-1000 ultraviolet crosslinker (UVP) at 120 mJ/cm². The membrane was then incubated with DIG Easy Hyb Granules (#11796895001, Roche) for 1 h in a hybridization oven at 50°C. A DIG-labeled probe prepared using the PCR DIG Probe Synthesis Kit (#11636090910, Roche) was added and incubated for >18 h, followed by two washes with wash buffer I (2× SSC, 0.1% SDS) for 15 min in a hybridization oven at 50°C, and an additional wash with wash buffer II (0.1× SSC, 0.1% SDS) for 15 min at 50°C. The membrane was then washed with 1× maleic acid buffer (100 mM maleic acid, 150 mM NaCl pH 7.0, adjusted with NaOH) for 10 min at room temperature and incubated with Blocking Reagent (#11096176001, Roche) for 30 min. Anti-Digoxigenin-AP, Fab fragments (#11093274910, Roche) was added to the Blocking Reagent, and the membrane was further incubated for 1 h. After that, the membrane was washed with wash buffer III (1× maleic acid buffer, 0.3% Tween 20) for 10 min three times, and equilibrated with equilibration buffer (100 mM Tris–HCl, 100 mM NaCl pH 9.5). The membrane was reacted with CDP-star (#11759051001, Roche) for 10 min, and chemiluminescence was detected with the LAS-4000 system (GE Healthcare). Relative RNA levels were determined using Multi Gauge v3.0 (Fujifilm, Tokyo, Japan).

Trichloroacetic acid (TCA) precipitation for protein preparation

The yeast cell pellet in a 1.5 ml tube (on ice) was resuspended with 500 μl ice-cold TCA buffer (20 mM Tris–HCl pH 8.0, 50 mM NH₄OAc, 2 mM EDTA and 1 mM PMSF) and transferred to a new 1.5 ml tube containing 500 μl of 20% TCA and 500 μl of 0.5 mm Zirconia/Silica Beads (BioSpec Products, Bartlesville, OK, USA). The cells were vortexed for 30 s three times, and the supernatant was transferred to a new 1.5 ml tube. Another 500 μl ice-cold TCA buffer was added to a beads-containing 1.5 ml tube, vortexed for 30 s, and then the mixture of supernatant and TCA buffer was transferred to a 1.5 ml tube. After centrifugation of lysates (14 000 rpm, 15 min, 4°C), the supernatant was discarded, and the pellet was dissolved in SDS sample buffer (125 mM Tris–HCl pH 6.8, 4% SDS, 20% glycerol, 100 mM DTT, 0.01% bromophenol blue; 150 μl/6OD₆₀₀) and heated at 95°C for 5 min (65°C for 15 min in Figure 2A). The protein solution was used for SDS-PAGE.

Electrophoresis and western blotting

Protein samples were separated by SDS-PAGE with 12% polyacrylamide gel in 50 mM-Tris, 400 mM glycine, 0.2% SDS buffer condition, then analysed by CBB staining or transferred onto PVDF membranes (Immobilon-P, Merck Millipore, MA, USA). Membranes were blocked with 5% skim milk in PBST (10 mM Na₂HPO₄/NaH₂PO₄ pH 7.5, 0.9% NaCl and 0.1% Tween 20), incubated with primary antibodies for 1 h at room temperature, washed three times in PBST, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. For HA-tagged protein detection, the membrane was incubated with HRP-conjugated antibodies. After washing with PBST three times, chemiluminescence was detected with the LAS4000 system (GE Healthcare, Chicago, IL, USA). Primary antibodies used for western blotting were as follows: anti-HA-peroxidase was purchased from Roche (#12013819001, RRID: AB 390917); anti-FLAG M2 antibody was purchased from Sigma (# F1804-1MG, RRID: AB 262044).

In vitro translation of W(UGG)12 reporter mRNA and Hel2-mediated ubiquitination

W(UGG)12 reporter mRNA was produced using the mMessage mMachine Kit (Thermo Fischer, Waltham, MA, USA) and used in a yeast cell-free translation extract from uS10-3HA ski2Δ cells. After preparation of the yeast translation extract, in vitro translation was performed as described previously (12). The cells were grown in YPD medium to an OD₆₀₀ of 1.5–2.0, washed with water and 1% KCl, and finally incubated with 10 mM DTT in 100 mM Tris, pH 8.0, for 15 min at room temperature. To generate spheroplasts, 2.08 mg zymolyase per 1 g cell pellet was added to YPD medium with 1 M sorbitol and incubated for 75 min at 30°C. Spheroplasts were then washed three times with YPD/1 M sorbitol and once with 1 M sorbitol, and lysed as described previously (12) with a douncer in lysis buffer [20 mM HEPES pH 7.5, 100 mM KOAc, 2 mM Mg(OAc)₂, 10% glycerol, 1 mM DTT, 0.5 mM PMSF and complete EDTA-free protease inhibitors (GE Healthcare)]. An S100 fraction was obtained from the lysate by low-speed centrifugation followed by ultracentrifugation of the supernatant. The S100 was passed through a PD10 column (GE Healthcare). In vitro translation was performed at 17°C for 60 min using an excess of template mRNA (38 μg per 415 μl extract) to prevent degradation of the resulting stalled ribosomes by endogenous response factors. For Hel2-supplemented in vitro translation, 32 nM Hel2 was added to the reaction. Hel2 purified from a yeast strain overexpressing Hel2-Flag was cultured in SC medium as described previously (12).

Sucrose density gradient centrifugation (SDG)

Yeast cells were grown exponentially at 30°C and treated with 0.1 mg/ml cycloheximide for 5 min before harvesting. Cells were harvested by centrifugation, and the cell pellet was frozen and ground in liquid nitrogen using a mortar. The cell powder was resuspended with lysis buffer [20 mM HEPES–KOH pH 7.4, 100 mM potassium acetate, 2 mM magnesium acetate, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride and 1 tablet/10 ml complete mini EDTA-free (#11836170001, Roche)] to prepare the crude extracts. Sucrose gradients (10–50% sucrose in 10 mM Tris-acetate pH 7.4, 70 mM ammonium acetate and 4 mM magnesium acetate) were prepared in 25  ×  89 mm polyallomer tubes (Beckman Coulter) using a Gradient Master (BioComp). Crude extracts (the equivalent of 50 A₂₆₀ units) were layered on top of the sucrose gradients and centrifuged at 150 000 × g in a P28S rotor (Hitachi Koki, Japan) for 2.5 h at 4°C. The gradients were then fractionated with BioComp Piston Gradient Fractionator. The polysome profiles were generated by continuous absorbance measurement at 254 nm using a single path UV-1 optical unit (ATTO Biomini UV-monitor) connected to a chart recorder (ATTO digital mini-recorder). For the western blots shown in Figures 2F and 4C, 900 μl of each fraction was mixed with 180 μl of 100% TCA and incubated for 15 min at 4°C. After centrifugation (14 000 rpm, 15 min, 4°C), the supernatant was removed, and the pellet was dissolved in SDS sample buffer (125 mM Tris–HCl pH 6.8, 4% SDS, 20% glycerol, 100 mM DTT, 0.01% bromophenol blue, 150 μl/6OD₆₀₀) and heated at 65°C for 15 min. The resulting solution was separated by SDS-PAGE.

Quantification and statistical analysis

The levels of RNA were quantified using Multi Gauge v3.0 (Fujifilm). All blot experiments were repeated three times independently, and a representative result is shown. Quantitative data are presented as the mean ± standard deviation (SD) from at least three independent experiments.

Consecutive tryptophan codons induce RQC2-independent RQC

We previously demonstrated that consecutive tryptophan codons induce translation arrest leading to proteasomal degradation (32). The E3 ubiquitin ligase Ltn1 catalyses the ubiquitination of the arrested products derived from the R(CGN)12 reporter on the 60S subunit, thereby targeting them for degradation by the proteasome. In this study, we detected arrest products derived from the GFP-W(UGG)12-HIS3 reporter compared with the GFP-R(CGN)12-HIS3 reporter (Figure 1A). Arrest products derived from the W(UGG)12 reporter were detected in ltn1Δ cells, indicating that the 12 consecutive tryptophan codons induce RQC (Figure 1A). The GFP-W12-FS-HIS3 reporter is identical to the GFP-W12-HIS3 reporter except for an additional nucleotide just before the sequence encoding polytryptophan and two additional nucleotides following the sequence. This frameshift mutation eliminated the arrest products, indicating that the consecutive tryptophan amino acid sequence, and not the nucleotide sequence, is necessary for inducing RQC. It could not be excluded that a frameshift abolishes the pairing of mRNA and tRNA, as well as change the amino acid sequence, and may lead to the effect.

Figure 1.

Consecutive tryptophan codons induce Rqc2-independent RQC. (A) Consecutive basic amino acids or tryptophan sequences induce ribosome-associated quality control (RQC). Cells harbouring the indicated pGPDp-GFP-X-HIS3 plasmid were grown in SC-Ura medium, and the samples were analysed by western blotting with anti-GFP or anti-EF3 antibody. (B) Rqt1/Hel2-dependent ubiquitination of uS10 and of the RQC-trigger (RQT) complex is required for RQC induced by consecutive tryptophan codons. The protein samples were prepared from the indicated mutant cells containing the GFP-W(UGG)12-HIS3 reporter and were subjected to western blot analysis. (C) Schematic drawing of the L4-W(UGG)12 transcript used in the in vitro translation (IVT) assay (upper panel). Scheme of ribosome-nascent polypeptide complex (RNC) purification (lower panel). (D) RNCs with L4-W(UGG)12 were prepared by IVT with addition of purified Hel2, separated by sucrose density gradient and detected by UV absorbance at 254 nm wavelength. Tagged uS10 in each fraction was detected by immunoblotting using anti-HA antibody. (E) Mutational analysis of 25S rRNA. The indicated 25S rRNA mutant variants were expressed from a plasmid in an rDNA deletion strain (NOY986)(12). Protein samples were prepared from the indicated cells containing HA-SDD1, HA-GFP-W12-HIS3 and HA-GFP-R12-HIS3 plasmids, and were analysed by western blotting. (F) The levels of the arrest products derived from GFP-W(UGG)12-HIS3 were decreased in the uL4 mutant cells. The indicated uL4 alleles were expressed from plasmids in the uL4Δ (uL4AΔuL4BΔ) strain (12).

The E3 ubiquitin ligase Hel2 is involved in translation arrest induced by polyarginine sequences. The W(UGG)12 reporter assay showed that arrest products were eliminated in the hel2Δltn1Δ double mutants, indicating that RQC induced by polytryptophan requires the E3 ligase as well as R(CGN)12 (Figure 1A, lanes 3, 6 and 9). We previously reported that the RQT complex, composed of the RNA helicase-family protein Slh1, the ubiquitin-binding protein Cue3 and Rqt4, is required for RQC induced by the R(CGN)12 reporter (5,12). To determine the involvement of the RQT complex in RQC induced by W(UGG)12 reporters, we measured the levels of arrest products in each deletion mutant (Figure 1B). Although arrest products were detected in single deletion mutants of cue3Δ and rqt4Δ, arrest products were not detected in the cue3Δrqt4Δ double mutant, indicating that the RQT complex is essential for RQC induced by consecutive tryptophan codons such as the R(CGN)12 sequence (Figure 1B, lanes 6–8). RQT is reported to be composed of three subunits, Slh1, Cue3 and Rtq4 in yeast (5,12), and the conserved function of mammalian homologous of the yeast subunits was demonstrated (13,14). RQC induced by R12 sequence also completely depends on Slh1. The arrest products were partially reduced in cue3Δ and rqt4Δ single mutants, but almost diminished in cue3Δrqt4Δ double mutant cells, indicating that Cue3 and Rqt4 may independently and redundantly contribute to RQC. In this study, RQC induced by W12 demonstrated the same dependency to the subunits of the RQT complex, suggesting that RQT-dependent subunit dissociation induced by R12 and W12 may be the same mechanism. To examine the ubiquitination of uS10 on the RQC pathway, a reporter assay was performed in the uS10-K6/8R mutant defective in uS10 ubiquitination. The results showed that arrest products were abolished in the uS10-K6/8R mutant, as observed in the hel2Δ mutant. This suggests that Hel2-dependent ubiquitination of uS10 at K6 and K8 is essential for the activation of RQC systems (Figure 1B, lanes 9 and 10).

Next, we performed ribosome stalling-coupled ubiquitination using an in vitro translation reaction as previously reported (10,12). To purify the colliding RNCs, we used a cell-free in vitro translation system with PA-His-L4-W(UGG)12 model mRNA, which contains consecutive tryptophan codons and a sequence coding for two N-terminal epitope tags: a His-tag and a PA-tag (Figure 1C). Ribosomes translating the W(UGG)12 reporter mRNA stalled and formed tri-ribosomes, and Hel2 preferentially ubiquitinated trisomes (Figure 1D). This result is consistent with previous results from our group obtained with an endogenous stalling sequence of SDD1 mRNA (12). The specific interaction between the nascent peptide and the stalled ribosome in the exit tunnel is also crucial to induce RQC by SDD1 (12). Juxtaposed on the tunnel side, the nucleotides A2401 (A2059 in Escherichia coli) and A2404 (A2062 in E. coli) were exposed near the nascent polypeptide (NC), and mutation of these bases to G and U, respectively, impaired RQC induction by the W12 stalling sequence (Figure 1E) as observed for SDD1 stalling (12). Mutation of the uL4 loop (⁶⁸GRAV⁷¹ to AAAA) suppressed translational arrest and RQC by the W12 sequence (Figure 1F). This indicates that the interaction between consecutive tryptophan codons and ribosome tunnel was crucial for stalling and RQC by W12 similar to SDD1 stalling (12).

The consecutive tryptophan codons inhibit CAT-tailing by CGA codon cluster

Contrary to RQC induced by tandem CGA codons (R12), translation arrest induced by polytryptophan sequences produces arrest products without CAT-tailing in ltn1Δ cells (Figure 1A, lanes 2 and 5). Rqc2 is required for efficient recruitment of Ltn1 to the 60S subunit (18–20, 27,28). Moreover, Rqc2 engages the stalled nascent chain and recruits tRNAs charged with alanine and threonine, suggesting that the 60S subunit catalyses CAT-tail formation in an unusual reaction. Poly-basic amino acids (R12) or tandem CGA codons such as R(CGA)4 induce translation arrest, and Rqc2-dependent CAT-tailing is observed in ltn1Δ cells (Figure 2A, lane 6; Figure 2B, lane 4). However, compared with RQC induced by R12 or R(CGA)4, translation arrest induced by polytryptophan sequences produces arrest products without CAT-tailing in ltn1Δ cells (Figure 2A, lane 2; Figure 2B, lane 8). In addition, arrest products are not detected in rqc2Δ single mutant cells in the W(UGG)12 reporter (Figure 2A, lane 3; Figure 2B, lane 6). Therefore, we propose that polytryptophan sequences induce Rqc2-independent RQC without CAT-tailing. A previous study demonstrated that the CAT-tail mediates the formation of SDS-resistant aggregates along with the polylysine tract present in nonstop proteins. In the R(CGN)12 reporter system, we confirmed the aggregation in ltn1Δ cells (Figure 2A, lane 6). However, in the W(UGG)12 reporter, this high molecular weight protein was not detected, indicating that the arrest products without the CAT-tail may not form aggregates (Figure 2A, lane 2).

Figure 2.

Consecutive tryptophan codons inhibit CAT-tailing by CGA codon cluster. (A) High molecular weight proteins expressed in ltn1Δ mutant cells are not detected in the W(UGG)12 reporter. (B) Consecutive tryptophan codons induce RQC without CAT-tailing. Cells harbouring pGPDp-GFP-R(CGA)4-HIS3 or pGPDp-GFP-W(UGG)12-HIS3 plasmids were grown in SC-Ura medium, and the samples were analysed by western blotting with anti-GFP or anti-EF3 antibody. (C) More than eight consecutive tryptophan residues located at the PTC inhibit CAT-tailing. (D) Consecutive tryptophan codons located near the PTC inhibit CGA codon-mediated CAT-tailing. (E) Consecutive tryptophan, but not phenylalanine or tyrosine sequences, inhibit CAT-tailing by R(CGA)4. (F) Model of the inhibition of CAT-tailing by consecutive tryptophan residues.

We hypothesised that consecutive tryptophan residues located at the peptidyl transferase center (PTC) or the discrimination gate of the ribosomal tunnel inhibit CAT-tailing. More than 11 consecutive tryptophan codons induced RQC without CAT-tailing (Figure 2B, lanes 7 and 8; Supplementary Figure S1). By contrast, the CGA codon cluster (CGA)4 efficiently induced RQC with CAT-tailing (Figure 2B, lane 4). We examined whether consecutive tryptophan residues inhibited CAT-tailing when translation arrest was induced by R(CGA)4 (Figure 2C). In GFP-R4-W6-HIS3, GFP-R4-W8-HIS3 and GFP-R4-W10-HIS3 reporters, polytryptophan sequences are not translated because translation arrest is induced by R4, and we observed the addition of CAT-tails to the arrest products (Figure 2C, lanes 5–10). However, GFP-W8-R4-HIS3 and GFP-W10-R4-HIS3 reporters produced arrest products without CAT-tailing (Figure 2C, lanes 13–16). In the GFP-W6-R4-HIS3 reporter, CAT-tailing was detected, suggesting that more than eight consecutive tryptophan residues located near the PTC inhibited CAT-tailing. We also examined the effects of the cluster of tryptophan residues before the CGA codons on RQC and CAT-tailing (Figure 2D). In the GFP-W7-R4-HIS3 reporter, CAT-tailing was detected, indicating that more than eight consecutive tryptophan residues located near the PTC inhibited CAT-tailing (Figure 2D, lanes 1–6; Figure 2E). We inserted CGA codons downstream of the eight consecutive tryptophan residues, and even a single CGA codon weakly induced RQC (Figure 2D, lanes 7–14). This is consistent with a composite stalling mechanism as demonstrated by AAA cluster and SDD1 stalling, in which the NC-induced interference with the PTC is combined with a difficult-to-decode mRNA for cooperative stalling (12). In the GFP-W8-R4-HIS3 construct, efficient ribosome stalling results in RQC2-independent of RQC without CAT-tailing (Figure 2D, lanes 13 and 14). The levels of arrest products with or without CAT-tailing derived from the indicated reporters in W303, ltn1Δ, rqc2Δ, ltn1Δrqc2Δ mutant cells were determined by western blot analysis (Figure 2E). The arrest products derived from these reporters were stabilized in ltn1Δ background. In RQC induced by W6-R4, polyphenylalanine (F10)-R4 and polytyrosine (Y10)-R4, the CAT-tailed arrest products were detected (Figure 2E). In contrast, the CAT-tailed arrest products were not detected in RQC by W10-R4, suggesting that F10 and Y10 sequences did not inhibit CAT-tailing induced by the R(CGA)4 sequence, suggesting that polyphenylalanine (F10) and polytyrosine (Y10) sequences did not inhibit CAT-tailing induced by the R(CGA)4 sequence (Figure 2E). We propose that polytryptophan sequences have a specific inhibitory effect on Rqc2-dependent CAT-tailing (Figure 2F).

Inhibition of CAT-tailing depends on the position of polytryptophan in peptidyl-tRNA

The above results demonstrate that more than eight consecutive tryptophan residues located near the PTC inhibit CAT-tailing. Next, we confirmed whether CAT-tailing inhibition is affected by the location of consecutive tryptophan residues. We constructed reporters by inserting consecutive glycine-serine (GS) sequences upstream or downstream of W(UGG)8, and 12 GS sequences were inserted upstream of R(CGA)4 to induce RQC (Figure 3). W(UGG)8 just upstream of R(CGA)4 efficiently inhibited CAT-tailing (Figure 3A, lanes 5 and 10), whereas the insertion of (GS)12, (GS)9, (GS)6, and (GS)3 between W(UGG)8 and R(CGA)4 abolished the inhibitory function of W(UGG)8 on CAT-tailing (Figure 3A, lanes 1–4 and 6–9). These results suggest that the position of W(UGG)8 relative to the PTC is crucial for inhibiting CAT-tailing. We then constructed GFP-W8-G-R(2–4)-HIS3 reporters containing a single glycine residue (G) between the CAT-tail-inhibitory element (W8) and the CAT-tail-inducing CGA codons [R(2–3)]. Two or three CGA codons downstream of eight consecutive tryptophan residues efficiently induced RQC (Figure 3B, lanes 1–2 and 5–6), and the insertion of a single glycine residue abolished the inhibitory effect of W(UGG)8 on CAT-tailing (Figure 3B, lanes 3–4, 7–8 and 11–12). We then aimed to identify the residue(s) between W(UGG)8 and R(CGA)4 critical for abolishing the inhibitory function of W(UGG)8 on CAT-tailing. To this end, we constructed the GFP-W8-X-R4-HIS3 reporter. Insertion of a single proline residue greatly diminished the inhibitory effect of W(UGG)8 on CAT-tailing (Figure 3C, lane 9; quantification in Figure 3E), and insertion of a single glycine residue moderately but significantly reduced the inhibitory effect of W(UGG)8 on CAT-tailing (Figure 3D, lane 12; Figure 3E). These results suggest that the inhibitory effect of W(UGG)8 on CAT-tailing depends on the position of polytryptophan in the peptidyl-tRNA at the P-site. Mass spectrometry of the arrest products derived from the R(CGN)12 reporter in ltn1Δ mutant cells revealed that RQC takes place when the ribosome translates the second and third CGA codons (5). Ribosome profiling also detected the accumulation of ribosome footprints in the second and third CGA codons (5). This suggests that translation of W(UGG)8-R(CGA)4 reporter mRNAs causes ribosome stalling at the second and third CGA codons, and peptidyl-tRNA containing consecutive arginine residues and eight tryptophan residues inhibits CAT-tailing in the 60S subunit (Figure 3F, left panel). Neither seven tryptophan residues nor the insertion of proline residues downstream of W(UGG)8 inhibited CAT-tailing in the 60S subunit, suggesting that the proper positioning of the eight tryptophan residues in the ribosome tunnel is crucial to inhibit CAT-tailing (Figure 3F, middle and right panels).

Figure 3.

Proper positioning of more than eight consecutive tryptophan codons on peptidyl-tRNA in the 60S subunit is required to inhibit CAT-tailing. (A) The insertion of GS residues between W(UGG)8 and R(CGA)4 abolished the inhibitory function of W(UGG)8 on CAT-tailing. The protein samples were prepared from W303ltn1Δ or W303ltn1Δrqc2Δ cells containing the indicated reporters. Western blot analysis was performed to evaluate the effect of glycine residue insertion on decreasing the inhibitory effect of W(UGG)8 on CAT-tailing. (B) The glycine residue between W(UGG)8 and R(CGA)2 or R(CGA)3 abolished the inhibitory effect of W(UGG)8 on CAT-tailing. The protein samples were prepared from W303ltn1Δ or W303ltn1Δrqc2Δ cells containing the indicated reporters. Western blot analysis was performed to evaluate the effect of glycine residue insertion on decreasing the inhibitory effect of W(UGG)8 on CAT-tailing. (C, D) Glycine and proline residue(s) between W(UGG)8 and R(CGA)4 abolished the inhibitory function of W(UGG)8 on CAT-tailing. The protein samples were prepared from W303ltn1Δ cells containing the GFP-W8-X-R4-HIS3 reporter. Western blot analysis was performed to evaluate the effect of the insertion of single residues on decreasing the inhibitory effect of W(UGG)8 on CAT-tailing. (E) Quantification of the efficiency of CAT-tailing in the reports shown in (C) and (D). The CAT-tailing efficiency was calculated as the ratio of [arrest products] + [CAT-tailed products] to [CAT-tailed products]. Three replicates were used to calculate the mean and SD. (F) Schematic drawing of nascent peptidyl-tRNA containing polytryptophan and arginine residues in the ribosome tunnel. The red circles indicate tryptophan residues, and open circles with R indicate arginine residues; the residue marked in black is Proline.

Consecutive tryptophan codons induce endonucleolytic cleavage (NGD)

The results of this study suggest that polytryptophan sequences induce translation arrest leading to proteasomal degradation. We therefore examined whether polytryptophan sequences also induce NGD, the mRNA surveillance pathway. To address this possibility, we detected intermediate endonucleolytic mRNA cleavage products, 5′ No-Go-Decay intermediate (5′-NGD-IM). The 5′-NGD-IM derived from arrest sequences is normally degraded by the exosome and can be detected in mutants lacking the exosome co-factor Ski2 (10,29). In ski2Δ mutant cells, 5′-NGD-IM derived from W(UGG)12 was detected in ski2Δ mutant cells (Figure 4A, lane 1), suggesting that consecutive tryptophan codons induced NGD. To determine the minimum number of tryptophan residues necessary for induction of NGD, we changed the number of consecutive tryptophan residues inserted between GFP and HIS3. The results showed that more than eight consecutive tryptophan codons induced NGD (Supplementary Figure S2B). The 3′-NGD-IM, which is normally degraded by the 5′-3′ exonuclease Xrn1, was detected in xrn1Δ mutant cells (Supplementary Figure S2C). In a previous study from our group, we reported that Hel2 is not only required for degradation of the aberrant peptide by the RQC system, but also for the decay of aberrant mRNA by the NGD pathway. Hel2-mediated ubiquitination of the ribosome is required both for canonical NGD (NGD^RQC+) and RQC coupled to the disome, and RQC-uncoupled NGD outside the disome (NGD^RQC−) can occur in a Not4-dependent manner (10). Consistently, 5′-NGD-IM was not detected in ski2Δhel2Δ mutant cells (Figure 4A). To obtain a more detailed insight into the sections of Hel2 involved in NGD and RQC, deletions were introduced into the Hel2 sequence. The 5′-NGD-IM and the peptide arrest products did not accumulate in the hel2 mutants carrying deletions of the RING domain (ΔRING) or alanine substitution of the conserved zinc-finger (ZnF) cysteine residues (C64A C67A) within the RING domain, indicating loss of function (Figure 4A, lanes 4 and 5). To determine whether K63-linked ubiquitin chains are critical for NGD, we used the ub-K63R mutant cell strain in which all endogenous ubiquitin-encoding genes are modified and only the K63R mutant ubiquitin was expressed (10). The level of 5′-NGD-IM derived from the R(CGN)₁₂ reporter mRNA was measured in an isogenic UB-WT strain with ski2 deletion and in ubiquitin mutant ski2Δub-K63R cells. The level of 5′-NGD-IM was significantly decreased in ski2Δub-K63R mutant cells (Figure 4B), indicating that endonucleolytic cleavage of mRNA in NGD required K63-linked ubiquitination.

Figure 4.

Consecutive tryptophan codons induce no-go decay (NGD). (A, B) Consecutive tryptophan codons induce NGD in the Hel2-mediated uS10 ubiquitination-dependent manner. RNA samples were prepared from cells and analysed by Northern blotting with DIG-labelled GFP or SCR1 probes. (C) More than eight consecutive tryptophan codons induced NGD. (D) 5′-NGD-IMs derived from GFP-W(UGG)12-HIS3 were detected in the indicated mutant cells by northern blotting with a DIG-labeled GFP probe. (E) Not4 increased the level of 5′-NGD-IMs derived from GFP-W(UGG)12-HIS3 in uS10-K6/8R mutant cells. (F) Not4-mediated eS7 ubiquitination increased the level of 5′-NGD-IMs derived from GFP-W(UGG)12-HIS3 in slh1 mutant cells. (G) The proposed model for endoribonucleolytic cleavages induced by consecutive tryptophan codons-mediated ribosome stalling. The red arrowheads in the left panel indicate the putative endoribonucleolytic cleavage sites in wild type. The blue arrowheads in the right panel indicate the putative cleavage sites in the mutants defective in the uS10 ubiquitination (uS10-K6/8R) or Slh1, an essential factor for the RQT-mediated subunit dissociation (slh1). Based on the reconstitution with collided ribosomes on SDD1 mRNA, it has been demonstrated that the leading and stalling ribosome is ubiquitinated and dissociated into subunits by the RQT complex (12). eS7 ubiquitination by Not4 and Hel2 E3 ligases are required for NGD^RQC- (10). Although it is strongly suggested that the leading ribosome in the trisome formed by SDD1-stalling sequence is ubiquitinated and dissociated into the subunits by the RQT complex (12), it is still unknown which collided ribosomes are ubiquitinated in the UGG codons-inducing substrates. There the position and species of the ubiquitinated ribosomes are not demonstrated in the model.

We recently reported that slh1Δ or uS10-K6/8R mutant cells produce other endonucleolytic mRNA cleavage sites, which are located >45–51 nucleotides upstream of the normal cleavage site, as determined using the R(CGN)12 reporter assay (10). In the W(UGG)12 reporter assay, the cleavage site was shifted upstream in slh1Δ or uS10-K6/8R mutant cells (Figure 4C, lanes 6, 8, 10, 12, 14, 16, 18 and 20; Supplementary Figure S2C). This shift was also observed in xrn1Δ mutant cells (Supplementary Figure S2D). Therefore, consecutive tryptophan sequences induce NGD via the same pathway as R(CGN)12, with RQC-coupled NGD (NGDRQC+) and RQC-uncoupled NGD (NGDRQC–) happening.

Not4-dependent ubiquitination is required for NGD^RQC− outside the disome (10). We therefore examined whether Not4-dependent ubiquitination of eS7A is required for NGD^RQC− by W12 in RQC-defective slh1/rqt2Δ or uS10-K6/8R mutant cells. The level of the shorter 5′-NGD-IM derived from the W(UGG)₁₂ reporter was slightly lower in ski2Δslh1Δnot4Δ cells than in ski2Δslh1Δ cells (Figure 4D, lanes 3 and 5), and the shorter 5′-NGD-IMs were also reduced in ski2Δnot4ΔuS10-K6/8R cells compared with the levels in ski2ΔuS10-K6/8R cells (Figure 4E; lanes 3 and 4, respectively). These results indicate that Not4 plays an important role in NGD^RQC− induced by W(UGG)₁₂. We then determined the levels and size of 5′-NGD-IMs derived from the W(UGG)₁₂ reporter in ski2ΔeS7AΔeS7BΔ or ski2Δslh1ΔeS7AΔeS7BΔ mutant cells harbouring plasmids expressing either eS7A wild-type (WT) or eS7A-4KR mutant (4KR). The shorter 5′-NGD-IM in slh1ΔeS7A-WT was produced by NGD^RQC−, and its level was higher than that produced by NGD^RQC+ in eS7A-WT cells (Figure 4F, lanes 1 and 2). The level of the shorter 5′-NGD-IM was lower in the slh1ΔeS7A-4KR mutant than in slh1ΔeS7A-WT (Figure 4F, lanes 2 and 4), confirming that Not4-mediated ubiquitination of eS7A plays a crucial role in endonucleolytic cleavage. Taken together, these results suggest that similar to NGD by R(CGN)₁₂, endonucleolytic cleavage by W(UGG)₁₂ in NGD^RQC− outside the disome requires stepwise ubiquitination of eS7A by the E3 ligases Not4 and Hel2 (Figure 4G).