Abstract
Selenocysteine-containing proteins play a central role in redox homeostasis. Their translation is a highly regulated process and is dependent on two tRNASec isodecoders differing by a single 2′-O-ribose methylation called Um34. Here we characterized FTSJ1 as the Um34 methyltransferase and show that its activity is required for efficient selenocysteine insertion at the UGA stop codon during translation. Specifically, loss of Um34 leads to ribosomal stalling and decreased UGA recoding. FTSJ1-deficient cells are more sensitive to oxidative stress and show decreased metastatic colonization in xenograft models of melanoma metastasis. We found that FTSJ1 mediates efficient translation of selenoproteins essential for the cellular antioxidant response. Our findings uncover a role for tRNASec Um34 modification in oxidative stress resistance and highlight FTSJ1 as a potential therapeutic target specific for metastatic disease.
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Data availability
Ribosomal-sequencing and RNA-sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE270971 and GSE270976. MS proteomics data and nucleoside modification analysis data have been provided as Supplementary Tables. All other data supporting the findings of this study are available from the corresponding author on reasonable request by email. Requests will be processed within 30 days. Source data are provided with this paper.
Code availability
All custom scripts used in this study are deposited at https://github.com/abcwcm/piskounova_ribo.
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Acknowledgements
This work was supported by the American Cancer Society (E.P.), an Elsa U. Pardee Foundation Grant (E.P.) and a Feldstein Medical Research Foundation Award (E.P.). L.A.N. is supported by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award.
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L.A.N. designed and performed experiments, analyzed data and wrote the paper. K.P.C., I.D., M.Z., G.C., R.O.H. and K.N.A. performed experiments and analyzed data. L.E.D. supervised CRISPR–Cas9 experiments and provided reagents. S.M. and S.J. designed, performed and supervised the ribosomal-sequencing experiments. M.A., P.Z. and D.B. designed, performed and supervised ribosomal-sequencing and RNA-sequencing data analysis. E.P. designed, performed and supervised experiments, analyzed data, supervised the data analysis and wrote the paper.
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L.E.D. is an advisor and holds equity in Mirimus, Inc., and is a consultant for Volastra Therapeutics and Frazier Healthcare Partners. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Extracellular selenium drives translation of selenoproteins.
(A) Levels and purity of tRNASec analyzed by SDS-PAGE gel after biotinylated pull-down from increasing amounts of small RNA input fraction. Representative image of 3 independent experiments shown. (B) Western blot analysis of selenocysteine proteins in DMEM/F12 media (0 nM Se) with increasing amounts of selenium in A375 cells. Representative image of 2 independent experiments shown. (C) Transcript levels of selenocysteine proteins in DMEM/F12 media (0 nM Se) with increasing amounts of selenium in A375 cells measured by qRT-PCR. Mean +/- SD. n = 3 independent replicates. Statistical significance assessed by two-way analysis of variance (ANOVA) with each condition compared to the zero selenium condition.
Extended Data Fig. 2 FTSJ1 is necessary and sufficient for Um34 modification on tRNASec.
(A) Co-immunoprecipitation assay of the interaction between HA-tagged FTSJ1 and FLAG-tagged component of selenocysteine machinery, SECp43, compared to empty vector control co-expressed in A375 melanoma cells. Representative experiment of 3 independent experiments shown. (B) Levels of Phe tRNA bound to FLAG-tagged Renilla, wild type or A26P-mutant FTSJ1, ALKBH8 and EEFSEC measured by qRT-PCR. n = 3. Mean +/- SD of 3 independent experiments. Two-sided unpaired t-test. (C) Lys UUU and Glu CUC tRNA levels bound to FLAG-tagged Renilla, wild type and A26P FTSJ1, ALKBH8 and EEFSEC by qRT-PCR. n = 3. Mean +/- SD of 3 independent experiments. (D) Quantification of Um34 levels on tRNASec in FTSJ1-KO SK-MEL-28 melanoma cells measured by LC-MS/MS. n = 6 from two independent experiments. Mean +/- SD. (E) Quantification of Um34 levels on tRNASec in FTSJ1-KO SK-MEL-2 melanoma cells measured by LC-MS/MS. n = 4 from two independent experiments. Mean +/- SD. (F) Quantification of Um34 levels in SK-MEL-28 melanoma cells overexpressing wild-type FTSJ1 or A26P-mutant. n = 3 independent experiments. Mean +/- SD. One-way analysis of variance (ANOVA) followed by Dunnett’s tests for multiple comparisons. (G) Quantification of Um34 modification levels on tRNASec in Control and FTSJ1-KO A375 melanoma cells overexpressing either Renilla luciferase, WT FTSJ1 or mutant A26P FTSJ1 measured by LC-MS/MS. n = 3-6 from two independent experiments. Mean +/- SD. Two-tailed one sample t-test. (H) Quantification of Um34 modification levels on tRNASec in Control and FTSJ1-KO HeLa cells overexpressing Renilla luciferase, WT FTSJ1 or mutant A26P FTSJ1 measured by LC-MS/MS. n = 2 independent experiments. Mean +/- SD. (I) Levels of recombinant FTSJ1 from a His- tag purification assessed by Coomassie staining. Representative of 2 independent experiments shown. (J) Levels of recombinant WDR6 from a His-tag purification assessed by Western blotting for WDR6. Representative of 2 independent experiments shown.
Extended Data Fig. 3 FTSJ1 regulates recoding of UGA as a selenocysteine.
(A) Global analysis of codon readthrough in FTSJ1-KO A375 melanoma cells compared to control A375 melanoma cells in the presence of selenium (30 nM). Pearson Correlation was used for statistical analysis, gray band is 95% confidence interval. (B) Levels of selenoprotein transcripts in Control and FTSJ1-KO A375 and SK-MEL-2 melanoma cell lines as measured by qRT-PCR. Mean +/- SD from 3 independent replicates. Mean +/- SD. Two-way ANOVA followed by Fisher LSD test for multiple comparisons. (C) Western blot analysis of selenocysteine proteins TXNRD1, GPX1 and GPX4 in normal (10% FBS), selenium-depleted (2%) and supplemented conditions (10% + 30 nM NaSe and 2% + 30 nM NaSe) in A375 melanoma cell line, HEK293Ts and SK-MEL-2 melanoma cell line. Representative of 3 independent experiments shown. (D) Transcript levels of TXNRD1, GPX4 and GPX1 in normal (10% FBS), selenium-depleted (2%) and supplemented conditions (10% + 30 nM NaSe and 2% + 30 nM NaSe) in A375 melanoma cell line by qRT-PCR. Mean +/- SD of 3 independent replicates. Two-sided unpaired t-test. (E) Western blot analysis of selenocysteine-containing proteins immunoprecipitated with selenocysteine-specific pulldown. SECISBP2 was used as a non-selenocysteine control protein. 1% of total protein input was run. Representative of 2 independent experiments shown. (F) Western blot analysis of selenocysteine-containing proteins immunoprecipitated with selenocysteine-specific pulldown from Control and FTSJ1-KO A375 melanoma cells. 1% of total protein input was run. (* = longer exposure of film) Representative of 3 independent experiments shown. (G) Western blot analysis of selenocysteine-containing proteins immunoprecipitated with selenocysteine-specific pulldown from A375 melanoma cells overexpressing Renilla or FTSJ1. 1% of total protein input was run. Representative of 2 independent experiments shown.
Extended Data Fig. 4 Loss of FTSJ1 causes ribosomal stalling around Sec UGA in a subset of selenoproteins.
(A) Cumulative RPF plots for affected selenoproteins in WT A375 melanoma cells in the absence (red) and presence (blue) of selenium (30 nM NaSe). (B) Cumulative RPF plots for affected selenoproteins in Control (blue) and FTSJ1-KO (red) melanoma cells in the presence of selenium (30 nM NaSe).
Extended Data Fig. 5 Loss of FTSJ1 has no additional effect on selenoprotein translation in the absence of selenium.
(A) Cumulative RPF plots for unaffected selenoproteins in WT A375 melanoma cells in the absence (red) and presence (blue) of selenium (30 nM NaSe). (B) Cumulative RPF plots for unaffected selenoproteins in Control (blue) and FTSJ1-KO (red) melanoma cells in the presence of selenium (30 nM NaSe).
Extended Data Fig. 6 Loss of FTSJ1 causes ribosomal stalling upstream of the Sec UGA in selenoproteins.
Non-cumulative RPF plots for all detected selenoproteins in Control or FTSJ1-KO A375 melanoma cells in the presence of selenium (30 nM NaSe).
Extended Data Fig. 7 Sensitivity to oxidative stress can not be rescued by overexpression of other FTSJ1 tRNA targets.
(A) Mitochondrial ROS levels measured by flow cytometry using CellROXGreen dye in Control and A375 melanoma cells in the presence of selenium (30 nM). n = 3 independent experiments. (B) Cytoplasmic ROS levels measured by flow cytometry using CellROX DeepRed in Control and A375 melanoma cells in the presence of selenium (30 nM). n = 3 independent experiments. (C) Lipid peroxidation levels measured by flow cytometry using BODIPY665/676 in Control and A375 melanoma cells in the presence of selenium (30 nM). Mean +/- SD. Representative of 2 independent experiments. (D-E) Cell survival of Control vs FTSJ1-KO A375 melanoma cells under increasing concentrations of RSL3 in the presence (D) or absence (E) of selenium. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). (F-G) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without FTSJ1 overexpression under increasing concentrations of RSL3 in the presence (F) or absence (G) of selenium. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). (H-I) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without FTSJ1 overexpression under increasing concentrations of RSL3 in the absence (H) or presence (I) of selenium. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). (J-K) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression of Phe tRNA with 25uM of pyocyanin in the presence (J) or absence (K) of selenium (30 nM NaSe). n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). (L) Quantification of Um34 modification levels on tRNASec in Control, WT tRNASec or C65G mutant tRNASec overexpressing A375 melanoma cells. n = 3 independent experiments. Mean +/- SD. Ordinary one-way ANOVA followed by Uncorrected Fisher’s LSD test for multiple comparisons. (M-N) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression of Phe tRNA with 25uM of pyocyanin in the presence (M) or absence (N) of selenium (30nM NaSe). n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). (O-P) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression of Trp tRNA with 25uM of pyocyanin in the presence (O) or absence (P) of selenium (30nM NaSe). n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided).
Extended Data Fig. 8 FTSJ1 overexpression can partially rescue sensitivity to oxidative stress while other tRNA targets of FTSJ1 can not.
(A) Cell survival of Control and FTSJ1-KO A375 melanoma cells with and without FTSJ1-overexpression under increasing concentrations of pyocyanin in the presence of selenium (30 nM NaSe) as measured by Cell Titer Glo. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). Representative of 3 independent experiments. (B) Cell survival of Control and FTSJ1-KO A375 melanoma cells with and without FTSJ1-overexpression under increasing concentrations of pyocyanin in the absence of selenium as measured by Cell Titer Glo. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). Representative of 3 independent experiments. (C) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression Phe tRNA under increasing concentrations of pyocyanin in the presence of selenium (30 nM NaSe). n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). Representative of 3 independent experiments. (D) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression Phe tRNA under increasing concentrations of pyocyanin in the absence of selenium. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). Representative of 3 independent experiments. (E) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression Trp tRNA under increasing concentrations of pyocyanin in the presence of selenium (30 nM NaSe). n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). Representative of 3 independent experiments. (F) Cell survival of Control or FTSJ1-KO A375 melanoma cells with or without overexpression Trp tRNA under increasing concentrations of pyocyanin in the absence of selenium. n = 3 independent culture experiments. Mean +/- SD. Representative of 3 independent experiments. Statistical difference between IC50 values analyzed by Extra-Sum-of-Squares F-Test (two-sided). Representative of 3 independent experiments.
Extended Data Fig. 9 Metastases show translational upregulation of selenoproteins.
(A) Transcript levels of selenoproteins in primary tumors compared to macro and micro metastatic nodules, isolated from n = 3 NSG mice transplanted with patient-derived melanomas M481 or M405, as measured by RNA-sequencing, quantified as a z-score. Table of p-values and log2 Fold Change listed in Appendix A. p-values were generated using DESeq2 algorithm (Wald test). Multiple comparisons adjustment was calculated using False Discovery Rate (FDR)/Benjamini-Hochberg test. (B) Levels of precursor tRNASec in Primary, macro and micrometastatic nodules from patient-derived M405 melanoma measured by qRT-PCR. Mean +/- SD from 3 independent replicates. One-way analysis of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons. (C) Levels of mature tRNASec in Primary, macro and micrometastatic nodules from patient-derived M405 melanoma measured by qRT-PCR. Mean +/- SD from 3 independent replicates. One-way analysis of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons. (D) Levels of gROSI variant levels with No UTR, GPX1 SECIS or GPX4 SECIS in primary and metastatic nodules, formed by A375 melanoma cells transplanted into NSG mice, as measured by immunofluorescence staining for GFP expression. n = number of mice analyzed, indicated in the figure. (E) Quantification of the GFP signal from gROSI variants shown in (E). Mean +/- SD. Two-way ANOVA followed by two-tailed Fisher’s LSD test.
Extended Data Fig. 10 Loss of FTSJ1 affects metastatic colonization but has no effect on lipid peroxidation levels.
(A) Western blot analysis of control and FTSJ1-KO A375 melanoma primary tumors. Representative from 3 independent experiments. (B) qRT-PCR analysis of FTSJ1-targeting shRNAs in A375 melanoma cells. Mean +/- SD from 3 independent replicates. One-way analysis of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons. (C) Western blot analysis of FTSJ1-targeting shRNAs in MeWo melanoma cells. Representative from 3 independent experiments. (D) Subcutaneous tumor growth of MeWo cells transplanted into mice expressing Control or shRNA5-targeting FTSJ1. n = 5 mice per group. Representative of 3 independent experiments. Mean +/- SD. (E-F) Western blot analysis of (E) M405 or (F) M481 tumors with either control or FTSJ1-targeting shRNAs. Representative from 4 independent experiments. (G-H) Western blot analysis of (G) A375 or (H) M481 melanoma tumors overexpressing Control Renilla luciferase or FTSJ1. Representative from 3 independent experiments. (I) Growth of subcutaneous tumors in mice transplanted from patient-derived melanoma, M405, overexpressing either Renilla luciferase or FTSJ1. n = 5 mice per group. Mean +/- SD. Representative of 2 independent experiments. (J) Total metastatic burden in visceral organs of mice transplanted with patient-derived melanoma, M405, overexpressing either Renilla luciferase or FTSJ1. n = number of mice analyzed, indicated in the figure. Mean +/- SD. Statistical significance assessed by one-way analysis of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons. (K) Total metastatic burden in visceral organs of mice transplanted intravenously with either Control or FTSJ1 KO A375 melanoma cells. n = number of mice analyzed, indicated in the figure. Mean +/- SD. Unpaired two-tailed t-test. (L) Total metastatic burden in visceral organs of mice transplanted intravenously with either Control or FTSJ1 overexpressing A375 melanoma cells. n = number of mice analyzed, indicated in the figure. Mean +/- SD. (M) Quantification of 4-HNE immunofluorescent staining of subcutaneous tumors expressing either control shRNA or FTSJ1-targeting shRNAs. n = 3 mice, each replicate is a tumor. Mean +/- SD. Ordinary one-way ANOVA, multiple comparisons calculated using uncorrected Fisher’s LSD test. (N) Flow cytometry analysis of lipid peroxidation levels in subcutaneous tumors overexpressing Control Renilla luciferase or FTSJ1, using BODIPY dye. n = 10 mice per group. Unpaired two-tailed t-test.
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Nease, L.A., Church, K.P., Delclaux, I. et al. Selenocysteine tRNA methylation promotes oxidative stress resistance in melanoma metastasis. Nat Cancer 5, 1868–1884 (2024). https://doi.org/10.1038/s43018-024-00844-8
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DOI: https://doi.org/10.1038/s43018-024-00844-8
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