Abstract
The human genome contains 61 codons encoding 20 amino acids. Synonymous codons representing a given amino acid are decoded by a set of transfer RNAs (tRNAs) called isoacceptors. We report the surprising observation that two isoacceptor tRNAs that decode synonymous codons become modulated in opposing directions during breast cancer progression. Specifically, tRNAIleUAU became upregulated, whereas tRNAIleGAU became repressed as breast cancer cells attained enhanced metastatic capacity. Functionally, tRNAIleUAU promoted and tRNAIleGAU suppressed metastatic colonization in mouse xenograft models. These tRNAs mediated opposing effects on codon-dependent translation of growth-promoting genes, consistent with genomic enrichment or depletion of their cognate codons in mitotic genes. Our findings uncover a specific isoacceptor tRNA pair that act in opposition, divergently impacting growth-regulating genes and a disease phenotype. Degeneracy of the genetic code can thus be biologically exploited by human cancer cells via tRNA isoacceptor shifts that causally facilitate the transition toward a growth-promoting state.
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Data availability
Raw sequencing data for ChIP, ribosomal profiling and polysomal profiling experiments that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession numbers GSE140957, GSE72349 and GSE172354. Mass spectrometry data are deposited in the Proteomics Identifications Database under accession number PXD036670. Previously published deep-sequencing data used in this study include GSE77401. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.
Code availability
Coding scripts used will be available from the corresponding author upon request. Source data are provided with this paper.
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Acknowledgements
We thank members of the laboratory of S.F.T. and H. Zaher for thoughtful comments on previous versions of the manuscript. We thank A. Ashbrook and the laboratory of C. Rice at The Rockefeller University for technical assistance with polysome profiling. We also thank The Rockefeller University resource centers, A. North and staff at the BioImaging Resource Facility, C. Zhao from the Genomics Resource Center, S. Heissel and H. Molina from the Proteomics Resource Center, V. Francis from the Comparative Bioscience Center and veterinary staff for animal husbandry and care. We also acknowledge A. Navickas, K. Garcia and D. Ruggero for help with the polysome profiling measurements. L.B.E.-N. and M.C.P. were supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD/PhD program. D.H. was supported by a National Institutes of Health Institutional Service Award to Memorial Sloan Kettering under award number 2T32CA009512-29A1 and The Damon Runyon Cancer Research Foundation. H.G. was supported by R00CA194077 and R01CA24098. S.F.T. was supported by the Breast Cancer Research Foundation award, the Reem-Kayden award, NCI grant R01CA215491 and an HHMI Faculty Scholar Award. S.F.T. and the laboratory of S.F.T. were supported by the Black Family Metastasis Research Center Award and the Emerald Foundation, an NCI U54CA261701 award, an NCI R01CA236954 award and an NCI R35CA274446 award.
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L.B.E.-N., H.G. and S.F.T. designed the experiments. L.B.E.-N., S.C., M.N. and M.C.P. performed the experiments. H.G. and H.A. performed ChIP–seq, ribosomal profiling, polysome profiling sequencing and ribosomal dwelling time computational analyses. D.H. performed iPAGE codon analyses. L.B.E.-N. and S.F.T. wrote the paper with input from the coauthors.
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Extended data
Extended Data Fig. 1 POLR3A ChIP sequencing reveals differential occupancy in isogenic poorly and highly metastatic breast cancer pairs.
(a) tRNA genomic loci abundance measured as percent (%) input using RT-qPCR of POLR3A IP cDNA with GAPDH as a negative control; n = 3 biological replicates. (b) Motif analysis of POLR3A IP normalized to Input sequences using FIRE analysis. (c) Scatter plot showing the Polymerase III occupancy in MDA LM2 vs. Parental cells (x-axis) versus the relative tRNA expression in MDA LM2 vs. parental MDA cells by tRNA Capture–sequencing (y-axis); each dot represents an individual tRNA. (d) Quantification of lung metastatic nodules post extraction after tail vein injection of 1.5 × 105 HCC1806 Parental (n = 4) or highly metastatic derivatives LM2B (n = 3) or LM2C (n = 3), with representative histology. Scale bars represent 5 mm. (e) Northern blot quantification of tRNAIleUAU relative to U6 of two independently derived LM2 lines relative to HCC1806 Parental cells with representative blot; n = 6 biological replicates. (f) Relative genomic copy number of tRNAIle loci, quantified by RT-qPCR; n = 7 biological replicates for all loci except n = 6 for tRNAIleUAU 6.63, 19.15 & tRNAIleGAU X.6 loci for MDA Parental, n = 5 for tRNAIleUAU 6.63, 19.15 loci and n = 4 for tRNAIleGAU X.6 loci for MDA LM2. (g) Relative pre-tRNA abundance of tRNAIleAAU across multiple primers covering distinct genetic loci using RT-qPCR of MDA LM2 vs. MDA-MB-231; n = 4 biological replicates for all loci except n = 6 for tRNAIleAAU 17.34 for each cohort and tRNAIleAAU 6.59 for MDA LM2, n = 9 for tRNAIleAAU 6.59 for MDA Parental. (h) Schematic of human tRNAIleGAU and tRNAIleAAU sequences, followed by LNA probe sequence used to hybridize tRNAIleGAU. Blue boxes highlight sequence differences between tRNAIleGAU and tRNAIleAAU, and * indicate locked nucleic acid bases incorporated into the design of LNA probe to increase specificity during hybridization. Lower case bases represent standard reduced prediction scores of base identity. Two-sided un-paired student’s t-tests performed in 1a, d-g. All data is represented as mean values with +/− SEM.
Extended Data Fig. 2 tRNAIleUAU and tRNAIleGAU levels can be manipulated exogenously.
(a, b) Northern blot quantification of tRNAIleUAU relative to U6 of MDA (a) or HCC1806 (b) Parental cells with control or overexpression of tRNAIleUAU. N = 9, 5 biological replicates, respectively. (c) Same as (a) of LM2 cells depleted of tRNAIleUAU via CRISPR with Guide 1 or 2 versus control. n = 5 biological replicates except n = 4 for MDA LM2 depleted tRNAIleUAU with CRISPR Guide 2. (d) Estimated indels as a fraction from CRISPR guides targets tRNAIleUAU genetic loci in MDA-LM2 CRISPR cells relative to control. (e) Sanger sequence tracing of Ile TAT 6.29 loci for CRISPR guide 97 (1) targeting tRNAIleUAU and control. (f) tRNAIleGAU quantification by specific tRNAIleGAU probe RT-qPCR normalized to 18S probes of LM2 cells with control or overexpression of tRNAIleGAU. n = 9 biological replicates. (g, h) tRNAIleGAU quantification by specific tRNAIleGAU probe RT-qPCR normalized to 18S MDA Parental CRISPRi cells with guides targeting either control or tRNAIleGAU (g) or or shRNA targeting control or tRNAIleGAU (h). n = 5 biological replicates except n = 4 for MDA shRNA targeting tRNAIleGAU. (i) Mature tRNAIleGAU, tRNAIleUAU, and tRNAIleAAU quantification by specific tRNAIle probe RT-qPCR normalize to 18 S of MDA cells with shRNA targeting either control or tRNAIleGAU. n = 5 biological replicates. (j) pre-tRNAIle quantification by specific tRNAIleGAU (left) or tRNAIleUAU (right) probe RT-qPCR normalized to 18S probes for MDA cells with tRNAIleGAU depleted via CRISPRi and tRNAIleUAU overexpression or control. n = 4-5, and 7 biological replicates, respectively. (k) tRNAIle quantification by specific tRNAIleGAU (left) or pre-tRNAIleUAU (right) probe RT-qPCR normalized to 18 S for HCC1806 cells with tRNAIleGAU depleted and tRNAIleUAU overexpression or control. N = 7 (left) and 5 (right) biological replicates, respectively. (l) tRNAIle quantification by specific tRNAIleGAU (left) or pre-tRNAIleUAU (right) probe RT-qPCR normalized to 18S for MDA MB 468 cells with tRNAIleGAU depleted and tRNAIleUAU overexpression or control. n = 4 biological replicates except n = 3 for tRNAIleGAU (left) quantification for tRNAIle modulated cells. (m) Primary tumor growth of NSG mice injected with either 1 × 106 MDA MB 231 control or tRNAIleGAU depletion tRNAIleUAU overexpression cells in one mammary fat pad, volume (mm3), n = 5 mice. (n) Bioluminescent imaging of peripheral blood collected from mice in (m) on Day 42, after 18 days in vitro culture. One sided (2a-c, f-g) or two sided (2h-l) un-paired student t-tests performed. All data is represented as mean values with +/− SEM.
Extended Data Fig. 3 Downstream effectors of tRNAIleGAU depletion and tRNAIleUAU overexpression mediate increased growth under metastatic stress conditions.
(a, b). Histogram of read coverage to demonstrate 3 nucleotide periodicity of the coding sequence with respect to the start (a) and stop codon (b) of the reading frame, representative images of 1 replicate. (c) Volcano plot representing log2 fold change vs. –log p value of translational efficiency from ribosomal profiling of MDA tRNAIleGAU depleted and tRNAIleUAU overexpression cells versus control. (d) Scatter plot representing the RNA vs. RPF log2 fold changes from ribosomal profiling of MDA tRNAIleGAU depleted and tRNAIleUAU overexpression cells versus control. (e) Relative cell counts of MDA MB 231 control & tRNAIleGAU depletion tRNAIleUAU overexpression cells after 5 days. N = 12 and 9 biological replicates, respectively. (f) iBAQ values of six candidate downstream effectors, measured by label free quantification mass spectrometry; 3 biological replicates each. (g) RT-qPCR quantification of LSM6 cDNA levels normalized to GAPDH on Day 2 of siRNA transfection, n = 3 biological replicates. (h) RT-qPCR quantification of PYCARD cDNA levels normalized to GAPDH; n = 3 biological replicates. (i) LICOR Western quantification of endogenous SMNDC1 levels in either MDA control or tRNAIleGAU depletion tRNAIleUAU overexpression cells with representative images, n = 3 biological replicates. (j) Relative mCherry expression levels in log2 format in MDA MB 231 tRNAIleGAU depleted tRNAIleUAU overexpression cells vs. MDA MB 231 control cells of an eGFP – split mCherry fluorescent reporter with either wildtype or codon mutant AUA → AUC PYCARD sequences. mCherry fluorescence intensity was measured by flow cytometry and normalized to GFP signals. N = 3 biological replicates. One (3i) or two-sided un-paired student’s t-tests were performed (3e, g, h). All data is represented as mean values with +/− SEM.
Extended Data Fig. 4 Ribosomal profiling of MDA cells concurrently modulated with tRNAIleGAU depletion & tRNAIleUAU overexpression.
(a) Polysome traces of two representative samples, measured by UV spectrometry. (b) mRNA levels of indicated genes GAPDH, SMNDC1, and EPHX1 in polysome profiling fractions in MDA MB 231 tRNAIleGAU depleted tRNAIleUAU overexpression cells versus control MDA MB 231 cells, with distinct expression levels in polysome enriched fractions. Experimental groups as labeled in (a), n = 2 biological replicates for control and n = 3 for tRNAIle modulated cells. 2-way ANOVA was performed, data is represented as mean values with +/− SEM. (c) Genes with a high abundance of AUA codons were significantly enriched among genes significantly upregulated in ribosomal protected fragments (corrected for their transcript changes) in tRNAIleGAU depleted tRNAIleUAU overexpression cells versus control MDA MB 231 cells, measured by ribosomal profiling. The statistical significance of these enrichments was assessed using mutual-information calculations and associated Z score (based on randomized input vectors). Also included is the χ2 p value for the associated contingency table. The heatmap was generated using the –log of the hypergeometric p value for enrichment and log of p value for depletion (collectively termed the enrichment score). The red and dark-blue borders indicate the statistical significance of the calculated hypergeometric p values. (d) Same as (c) except analyzed for AUC codon enrichment. Codon content scored for by relative synonymous codon usage score (RSCU).
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Earnest-Noble, L.B., Hsu, D., Chen, S. et al. Two isoleucyl tRNAs that decode synonymous codons divergently regulate breast cancer metastatic growth by controlling translation of proliferation-regulating genes. Nat Cancer 3, 1484–1497 (2022). https://doi.org/10.1038/s43018-022-00469-9
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DOI: https://doi.org/10.1038/s43018-022-00469-9
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