Ribosome Collision Is Critical for Quality Control during No-Go Decay Carrie L. Simms, Liewei L. Yan, Hani S. Zaher  Molecular Cell  Volume 68, Issue 2, Pages 361-373.e5 (October 2017) DOI: 10.1016/j.molcel.2017.08.019 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 NGD Reporters Produce Heterogeneous 5′ and 3′ Fragments (A) Northern analysis of 5′ fragment accumulation from the indicated PGK1 mRNA reporters in wild-type and ski2Δ strains. PGK1 indicates no additional sequence was added to the PGK1 ORF. SL indicates a stem loop was inserted at position 1040 of the ORF; (CGA)12, (AAA)12, and (UUU)12 indicate the corresponding codons were inserted at position 950 of the ORF. (B) Northern analysis of 3′ fragment accumulation in wild-type and xrn1Δ strains. The fragments were labeled as per Chen et al. (2010). Note that the asterisk depicts an endogenous endonucleolytic cleavage specific for the PGK1 sequence. (C–F) Mapping sites of mRNA cleavage in cells expressing stalling reporters. The 5′ ends of 3′ fragments from xrn1Δ cells were mapped relative to the stall site (C) (data from Chen et al., 2010). (D–F) The 3′ ends of 5′ mRNA cleavage products from ski2Δ cells expressing the indicated reporter were mapped relative to the stall site. Data are binned to 25 nt increments. See also Figure S1. Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Large-Scale Sequencing Reveals Strong Periodicity of Cleavage Sites (A) Plot of sequencing reads of 3′ RACE products from ski2Δ cells expressing a (CGA)12 reporter. Each point represents one read, mapped relative to the stall site. Inset shows a wider view of the mapped data. (B) Plot of smoothed data from (A) reveals a strong periodicity in the location of cleavage sites. Data were smoothed by using a five-point quadratic polynomial. Peaks were assigned by taking the derivative of the smoothed data and finding the + to − inflection point. (C) Graph depicting the number of reads as mapped to the translation frame of the reporter. (D) Plot of the distance between peaks in (B); mean ± SD from eleven points is shown. See also Figure S2. Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Endonucleolytic Cleavage Is Not Efficient on Ribosomes that Stall within 175 nt of the Start Codon (A) Northern analysis of 5′ fragment accumulation in ski2Δ cells expressing a reporter with the SL located at the indicated positions (relative to the start codon), performed on formaldehyde-agarose (top) or denaturing PAGE (bottom). 5′ cleavage products are indicated by arrowheads. (B) Northern blot of RNA from xrn1Δ cells expressing the same set of reporters as in (A) performed on formaldehyde-agarose. 3′ cleavage products are indicated by arrowheads. (C) Polysome profile, ethidium bromide-stained agarose gel, and northern blot from ski2Δ cells expressing the indicated SL reporter. The band corresponding to endogenous PGK1 is labeled PGK1 end. Blots in all panels were probed with an oligo complementary to the 5′ UTR immediately upstream of the AUG. See also Figure S3. Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Deletion of RPL1B Reduces mRNA Cleavage Efficiency by Lowering Ribosome Density on mRNAs (A) Efficiency of mRNA cleavage is reduced in ski2Δ rpl1bΔ cells compared to ski2Δ cells expressing PGK1-SL. Cleavage activity is rescued by RPL1B expression from a plasmid (pAG426-rpl1b). Cleavage products were quantified relative to the PGK1 full-length reporter (top band on blot) and to SCR1 (bottom panel). Percent (%) abundance compared to wild-type is shown. (B) Quantification of ribosomal protein content from ribosomes for rpl1bΔ versus wild-type cells by mass spectrometry. Values are relative ion counts ± SD from three biological replicates. (C) Polysome profiles from wild-type and rpl1bΔ cells. (D) Western blots of cell lysates from wild-type and rpl1bΔ cells used to assess RPS9 levels. Quantification of RPS9 relative to PGK1 is shown in the lower panel. Value is mean ± SD from three biological replicates. Full blots are shown in Figure S3. (E) A phosphorimage of an SDS-PAGE gel used to follow the incorporation of 35S-Met in nascent proteins in wild-type and rpl1bΔ cells. Middle panel shows Coomassie-stained samples used to assess the steady-state levels of proteins. Bottom panel is a western blot of PGK1. Radiolabeled proteins from two independent biological samples were quantified relative to the steady-state levels of PGK1 and the resulting plot is shown to the right. Values are mean ± SD. (F) A dual luciferase reporter was used to assess readthrough on (CGA)4. Plot of normalized firefly luciferase relative to Renilla luciferase expression from a control reporter and one containing a (CGA)4 sequence in wild-type and rpl1bΔ cells. Values are mean ± SD for three biological replicates. (G) Western blot of cell lysates from wild-type and rpl1bΔ cells expressing the indicated PGK1 reporters. (H) Northern blot of RNA isolated from wild-type and rpl1bΔ cells expressing the indicated reporters. Cleavage products were quantified relative to the full PGK-stall reporter (top band) or to SCR1 (bottom panel). The band corresponding to endogenous PGK1 is labeled PGK1 end. See also Figure S4. Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Reducing Ribosome Density by Deletion of RPS28B or by Limiting Initiation Inhibits NGD (A) Northern analysis of 3′ fragment accumulation in ski2Δ cells expressing the indicated reporters and deleted for rpl1b, rps26b, or rps28b. Cleavage products were quantified relative to the full PGK-stall reporter or to SCR1. The band corresponding to endogenous PGK1 is labeled PGK1 end. (B) Polysome profiles from ski2Δ, ski2Δ rps26bΔ, and ski2Δ rps28bΔ cells. (C) Western blot used to assess reporter protein expression downstream of the indicated 5′ UTR for either control PGK or (CGA)12. Cells were grown in the presence of glucose (−) to suppress induction or in the presence of galactose (+) to induce reporter expression. (D) Northern blot analysis of mRNA cleavage from reporters containing longer 5′ UTRs. See also Figure S5. Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Ribosome Collision Is Accompanied by Ubiquitination of Ribosomal Protein RPS3 (A) Bar graph showing the relative transcript levels of the RPL42A and RPL42B genes to TAF10 as assessed by qRT-PCR. The values are the mean of three biological repeats with error bars depicting the SD. (B) Growth curves of the indicated strains in the presence of the specified cycloheximide concentrations. (C) Western blot analysis of RPS3 modification (assessed by anti-FLAG antibody) in the cycloheximide-sensitive, -mixed, and -resistant strains in the presence or absence of the antibiotic. Arrowheads point to bands that appear in the presence of cycloheximide. (D) Western blot analysis of anti-FLAG immunoprecipitated protein under conditions as in (C). Arrowheads point to bands that are enriched after cycloheximide treatment. (E) Western blot analysis used to explore the effect of Hel2 on the cycloheximide-dependent modification of RPS3 as well as appearance of ubiquitinated protein products in the cycloheximide-mixed strain. (F) Western blot analysis used to follow RPS3 modification and appearance of ubiquitinated protein products as a function of cycloheximide concentration. Antibiotic concentrations from left to right: 100, 33, 11, 3.7, 1.2, 0.41, 0.14, 0.045, 0.015, 0.0051, and 0.00 μg/mL. See also Figure S6. Molecular Cell 2017 68, 361-373.e5DOI: (10.1016/j.molcel.2017.08.019) Copyright © 2017 Elsevier Inc. Terms and Conditions