Mistranslating the genetic code with leucine in yeast and mammalian cells

Figures & data

Figure 1. Schematic of natural and synthetic tRNA^Leu variants.

Anticodon variants of tRNA^Leu (red highlight) are found in the human population at low allele frequencies (Table 1). The tRNA identity elements (orange) and anticodon (gold) sequences are highlighted. Two variants of human (A) tRNA^Leu_CAA provide routes to generate tRNAs that decode Trp (A35C) or Phe (C34G) codons, while one variant in the tRNA^Leu_AAG gene (G36A) produces a tRNA that also mistranslates Phe codons with Leu. (B) We used experiments in yeast to investigate Leu mis-incorporation across the entire genetic code. The yeast tRNA^Leu_UAA gene was used as a basis for our inducible construct to characterize growth of 64 different yeast strains, each expressing a tRNA^Leu with a different anticodon.

Table 1. Naturally occurring human tRNA^Leu variants characterized in this study.

tRNA Gene [33]	tRNA variant, dbSNP ID	Variant anticodon	codons read	AA mis-incorporated	minor allele frequency
Leu-CAA-3–1	A35C rs752934775	CCA	Trp UGG	Leu	0.03% [34]
Leu-CAA-3–1	C34G rs1032415115	GAA	Phe UUC/U	Leu	0.002–0.006% [35]
Leu-AAG-3–1	G36A rs1431919737	AAA	Phe UUU/C	Leu	0.0004% [32] − 0.007% [36]

Figure 2. Identification of Leu mis-incorporation in mammalian cells by tandem mass spectrometry.

LC-MS/MS analysis of a GFP-mCherry protein purified from N2a cells expressing (A,B) wild-type tRNA^Leu_CAA or one of the Trp- or Phe-decoding tRNA^Leu variants: (C, D) tRNA^Leu_CCA (A35C), (E, F) tRNA^Leu_GAA (C34G), (G, H) tRNA^Leu_AAA (G36A). The MS/MS data are summarized in (A, C, E, G) coverage maps showing confident peptide hits representing normal translation (blue lines) and Leu mis-incorporation (boxed L). Representative MS/MS spectra (B, D, F, H) demonstrating Leu mis-incorporation are shown and annotated with peptide quality scores (-10logP). Compared to normal cells, cells expressing the mutant tRNAs show more mistranslated peptide hits with higher quality scores, indicating a more confident match of the peptide to the observed spectra. Table 2 includes a complete summary of mistranslated peptides identified by MS/MS.

Table 2. Summary of Leu mis-incorporation identified by MS/MS in mammalian cells.

tRNA gene	tRNA variant	GFP-mCherry residue*	peptide hit maximal −10logP	ion counts (# peptide hits) Leu/Trp or Phe
Leu-CAA-3-1	WT	F338L	45.24	1 L/21 F
Leu-CAA-3-1	A35C	W309L	75.61	1 L/21 W
	A35C	W394L	41.44	2 L/7 W
Leu-CAA-3-1	C34G	F9L	85.97	2 L/21 F
	C34G	F28L	83.45	2 L/7 F
	C34G	F100L	58.73	1 L/4 F
	C34G	F101L	58.82	1 L/4 F
	C34G	F115L	51.51	1 L/30 F
	C34G	F278L	79.83	1 L/30 F
	C34G	F307L	76.58	2 L/16 F
	C34G	F338L	59.29	1 L/42 F
	C34G	F342L	46.13	1 L/41 F
	C34G	F350L	74.80	1 L/35 F
	C34G	F369L	72.06	3 L/47 F
Leu-AAG-3-1	G36A	F9L	86.76	3 L/23 F
	G36A	F28L	75.26	2 L/31 F
	G36A	F115L	47.65	1 L/30 F
	G36A	F338L	38.77	1 L/31 F
	G36A	F369L	80.67	4 L/46 F

*Residue numbering is based on the GFP-mCherry construct in the WT-PAN plasmid (Addgene plasmid #99638) [37]. All Phe codons are UUC in this construct.

Figure 3. OTTR-seq analysis of N2a cells expressing wild-type human tRNA or anticodon variants.

The level of each tRNA anticodon variant was plotted relative to (A) the level of the pool of the corresponding wild-type tRNA^Leu isodecoders and to (B) the level of the pool of competing tRNA^Trp or tRNA^Phe isodecoders. Statistical analysis was computed with a pairwise t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C, D) OTTR-seq identified mis-incorporation of G at the A34 site, indicating A-to-I editing at position 34. (C) In the total pool of tRNA^Leu_AAG, A34I levels were significantly reduced in cells expressing tRNA^Leu_AAA. (D) In cells transfected with tRNA^Leu_AAA, we plotted the proportion of reads with A34I that corresponded to the wild-type background (G36) or the mutant tRNA^Leu (A36), which showed nearly stoichiometric A34I in wild-type tRNA^Leu; in tRNA^Leu_AAA ~45% of the A34 residues were converted to I. (E) OTTR-seq mismatch coverage plots from cells expressing the indicated wild-type tRNA^Leu, Trp-, or Phe-decoding tRNA^Leu variant are shown at the resolution of individual bases. Nearly all reads shown are uniquely mapped or mapped within the isoacceptor group (Fig. S2D). Reads shown as grey bars indicate the level of the wild-type or expected tRNA sequence, while coloured bars indicate base mis-incorporation levels of A (green), T (red), C (blue), or G (orange). Base mis-incorporations reveal the level of the expressed human tRNA^Leu variants as well as base modification levels at the annotated loci. The human genes differ at position e9 (*e9) and position 57 (G57A) from the mouse background.

Figure 4. Global protein synthesis levels in cells expressing tRNA variants.

The puromycylation assay, based SUrface SEnsing of Translation (SUnSET) method [39], was used to quantified protein synthesis in N2a cells expressing tRNA^Leu or the indicated Trp or Phe-decoding variants. (A) Western blotting shows the level of puromycin incorporated in the proteome during a 30-min time course with vinculin used as a loading control. (B) Quantitation (B; N = 3 biological replicates) shows a significant defect in global protein synthesis in cells expressing the Phe-decoding tRNA^Leu_GAA. Statistical analysis was computed with a pairwise t-test (n.s. – not significant, * p < 0.05).

Figure 5. eGFP fluorescence and cytotoxicity in normal and mistranslating cells under proteasome inhibition.

N2a cells transfected with plasmids co-expressing eGFP-mCherry and wild-type tRNA^Leu_CAA or one of the indicated tRNA^Leu mutants. (A) Images were acquired with fluorescence and brightfield microscopy. (B) The mean eGFP fluorescence per cells was measured to assess protein production levels in normal and mistranslating cells. (C) Cytotoxicity was measured using the Cytotox-Glo live/dead cell assay. GFP fluorescence and relative cell death were measured under normal conditions (DMSO) and with the indicated concentrations of proteasome inhibitor (MG132). The data are based on N = 3 biological replicates. Error bars represent ± 1 standard deviation of the mean. Statistical analysis was computed with a pairwise t-test (n.s. – not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Bars indicate pairwise relationships for statistical significance; annotations above the data bar indicate statistical comparisons between each MG132 concentration and the DMSO condition for the same cells.

Figure 6. Impact of tRNA^Leu anticodon variants on yeast growth.

Representative growth curves of yeast strain CY8652 containing a plasmid with a control (A) tRNA^Leu_UAA(Leu), a (B) tRNA^Leu_UCA(Stop) variant and, a (C) tRNA^Leu_CAU(Met) variant. Blue, yellow and magenta lines represent strains grown in media containing 0, 0.01 and 1.0 μg/mL doxycycline, respectively. (D) Relative growth of all tRNA^Leu anticodon variants compared to control tRNA^Leu_UAA at 1.0 μg/mL doxycycline; raw data and statistical comparisons are in Supplementary Data Files 2 and 4). Cultures were grown for 48 h at 20°C in medium lacking uracil and leucine, diluted to an A₆₀₀ of 0.1 in the same medium with 1.0 μg/mL doxycycline and grown for 24 h at 30°C with agitation. A₆₀₀ was measured at 15-min intervals and doubling time was quantified with the ‘growthcurver’ R package. Each point represents one biological replicate (N = 4). The relative growth is calculated as the ratio of doubling time compared to tRNA^Leu_UAA with the exception of the CGG (Pro) anticodon where areas under the curve were compared. The relative growths for GCG, ACG (Arg), UUC (Glu), GCC and ACC (Gly) were designated at 0% as no transformants were obtained. (E) Relative growth of yeast strains expressing the indicated tRNA^Leu anticodon variants compared to wild-type tRNA^Leu_UAA at 0.01 μg/mL doxycycline (circles) and at 1.0 μg/mL doxycycline (triangles). The anticodon variants are ordered according to decreasing growth defect at 1.0 μg/mL doxycycline.

Figure 7. Identification of Leu mis-incorporation in yeast by tandem mass spectrometry.

(A-C) Mirror y- and b-ion plots of spectra for mistranslated (top) and wild-type peptide sequences (bottom). (A) Unique b14 (blue arrow) and y9 (red arrow) ions supporting Leu mis-incorporation at Phe codons are indicated in the spectrum and are not found in the spectrum from the wild-type peptide sequence. (B,C) Magnified images showing unique y- and b-ions supporting Leu-mis incorporation. Lower-case ‘f’ denotes the position of mis-incorporated Leu. (C) Box and whisker and (D) Log₁₀ plots of mistranslation frequency from label-free quantitation based on the area under the isotopic peak for each mis-translated peptide relative to the area under the isotopic peak for the accurately translated peptide. Individual data points (circles), mean (x), and standard deviation (error bars) are indicated. Statistical analysis was performed using ANOVA (* p < 0.05, *** p < 0.001). All peptides identified are listed in Supplementary Data File 3.

Figure 8. Relative growth of yeast strains with tRNA^Leu anticodon variants.

Growth of yeast strains with each tRNA^Leu anticodon variant was plotted relative to a yeast strain expressing wild-type tRNA^Leu_UAA. The data were overlaid on a circular representation of the genetic code. In this representation [116], more energetically stable codon-anticodon pairs (C or G at base 35 and 36) reside along the top of the chart, while less stable pairs (A or U 35 and 36) are located at the bottom. We observed a general trend where mistranslating tRNA^Leu variants with C or G at positions 35 or 36 tend to show greater phenotypic defects compared to tRNA^Leu variants with A or U at positions 35 or 36.

Figure 9. Analysis of anticodon variants in different contexts.

(A) Comparison of three tRNA^Leu isoacceptors, UAA (YNCB0012W), UAG (YNCJ0029W) and CAA (NCG0040C). Relative growth of transformants containing tRNA^Leu variants with AAA(Phe), CGU(Thr), and AAC(Val) anticodons in minimal media lacking leucine and uracil and containing 1.0 μg/mL doxycycline. The tRNA encoding genes were introduced into the tet^o plasmid identically but with their native flanking sequence and transformed in CY8652. The data for each anticodon mutant are compared to data from cells transformed with wild-type tRNA^Leu (relative growth = 1.00, dashed line). Error bars show ± 1 standard deviation based on three biological replicates. Statistical analysis was computed using ANOVA (** p < 0.01, *** p < 0.001) (B) Comparison of the relative growth of tRNA^Leu anticodon variants (triangles) and tRNA^Ala (circles). The graph is arranged from lowest to highest relative growth for the tRNA^Leu variants. tRNA^Leu variants that could not be transformed are indicated on the left at 0% relative growth. Anticodons for Leu and Ala are excluded. Relative growth for the tRNA^Leu variants is from Figure 6(e) and data for the tRNA^Ala variants is from Cozma et al. [65].

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Data availability statement

All data are available in the figures, tables, supplementary information, and Supplementary Data Files 1–4.

All MS/MS data files are available:

https://figshare.com/articles/dataset/Mass_spec_tRNALeu_2023_zip/23284907/1

https://bioinfor.sharefile.com/d-sb232430cb59d423c99b1cb5e3b492e58

OTTR-seq data files are available at NCBI BioProject database

http://www.ncbi.nlm.nih.gov/bioproject/1066329

SubmissionID:SUB14155213

BioProject ID: PRJNA1066329