Genome-wide Translation Profiling by Ribosome-Bound tRNA Capture Chien-Wen Chen, Motomasa Tanaka  Cell Reports  Volume 23, Issue 2, Pages 608-621 (April 2018) DOI: 10.1016/j.celrep.2018.03.035 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 23, 608-621DOI: (10.1016/j.celrep.2018.03.035) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Development of the Ribo-tRNA-Seq Technology (A) A scheme of simultaneous tRNA and mRNA ribosome profiling. Total RNA (tRNA-seq and mRNA-seq) and ribosome-bound tRNA and mRNA (Ribo-tRNA-seq, Ribo-mRNA-seq) were used for library preparation. (B) The correlation between tRNA anticodons from Ribo-tRNA-seq and E-, P-, A-site mRNA codons from Ribo-mRNA-seq. A comparison between Ribo-tRNA-seq and the pseudo P-site from mRNA-seq served as the control. Both mRNA and tRNA anticodons were converted to amino acids for direct comparison. n = 20, Pearson’s coefficient. (C) tRNA-seq detected various tRNA modifications in one tRNA gene. As an example, the termination and/or mismatch of RT reactions due to the presence of tRNA modifications allows one to detect modifications of m1G9, m22G26, I34, and M1I37 in Ala-tRNA-AGC. The positions of the change points were referred to tRNA modification database (Machnicka et al., 2013). (D–F) Validation of the presence of tRNA modifications. The tRNA modification-mediated alteration of RT reactions was recovered by the yeast deletion strains of trm1 (D), trm10 (E), and abp140 (F). (G) A summary of ten tRNA modifications identified by tRNA-seq and Ribo-tRNA-seq in this study. Three tRNA modifications validated by deletion of nonessential Trm1, Trm10, and Abp140 genes were denoted as black, while identification of the other tRNA modifications (red) are based on the tRNA database (Machnicka et al., 2013) and the fact that the modification moiety is involved in Watson-Crick hydrogen bonding required for RT reactions. See Table 1 for details. (H) Deletion of trm1-mediated m22G26 modification in tRNAs altered the tRNA population in m22G26-containing tRNAs, but not m22G26-lacking tRNAs, in tRNA-seq. Cell Reports 2018 23, 608-621DOI: (10.1016/j.celrep.2018.03.035) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Ribo-tRNA-Seq Is More Sensitive to Environmental Stress than tRNA-Seq Differences in the abundance of total (tRNA-seq) or ribosome-bound tRNAs (Ribo-tRNA-seq) between control and stress-induced cells. Color gradient represents relative amounts of tRNAs in stress-induced cells compared with those of control cells. Red and green color gradients show an increase and decrease of tRNA abundance, respectively, as compared with control. Cold, heat, sorbitol, copper, and H2O2 represent the 0°C cold shock, 42°C heat shock, 1 M sorbitol, 5 mM CuSO4, and 0.5 mM H2O2 treatment, respectively. The variation index represents the deviation of tRNA abundance from the averaged tRNA amount calculated from the cells under the five stressors. n ≥ 3. See Supplemental Experimental Procedures for details. Cell Reports 2018 23, 608-621DOI: (10.1016/j.celrep.2018.03.035) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Ribo-tRNA-Seq Serves as a Biosensor for Translation Control (A) Under amino acid starvation, the relative ratio of iMet-tRNA levels was increased in Ribo-tRNA-seq, but not in tRNA-seq (n = 47). (B) Ribo-mRNA-seq showed an increase in the frequency of ATG mRNA codon (red bar) at the P-site under amino acid starvation. Open and closed bars represent a relative population of mRNA codon under the normal and amino acid starvation conditions, respectively. (C) The iMet/18S ratio calculated from Ribo-tRNA-seq and tRNA-seq reveals an inhibitory mechanism of translation (see Supplemental Experimental Procedures for details). A dashed line (i.e., the ratio value 1) shows the border between initiation and elongation defects. Data represent mean + SEM (n ≥ 3). ∗∗∗p < 0.001 and ∗p < 0.05 by unpaired two-tailed Student’s t test. Cell Reports 2018 23, 608-621DOI: (10.1016/j.celrep.2018.03.035) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Partially 3′ CCA-Tail Degraded tRNAs Arrest Translation (A) Each tRNA species exhibited a different sensitivity of its 3′ CCA tail to degradation in distinct H2O2 concentrations. (B) Ribo-mRNA-seq showed a lower correlation of A-site mRNA codons between control and 2 mM H2O2-treated cells (R2 = 0.40) compared to P-site mRNA codons (R2 = 0.69) (n = 64). (C) The wobble pairing increased the association between A-site mRNA codons and ribosome-bound, CCA-tail-degraded tRNA anticodons upon 2 mM H2O2 treatment. Watson-Crick and wobble pairings of mRNA-tRNA codons are shown in open and closed bars, respectively. (D) At an amino acid level (n = 20), the product of a frequency of CCA-tail degradation in tRNA and a population of tRNA inside the ribosome showed a tendency toward a higher correlation with A-site mRNA codons (r = 0.62) than P-site mRNA codons (r = 0.38) in Ribo-mRNA-seq. Cell Reports 2018 23, 608-621DOI: (10.1016/j.celrep.2018.03.035) Copyright © 2018 The Authors Terms and Conditions