Two isoleucyl tRNAs that decode synonymous codons divergently regulate breast cancer metastatic growth by controlling translation of proliferation-regulating genes - PubMed (original) (raw)

Two isoleucyl tRNAs that decode synonymous codons divergently regulate breast cancer metastatic growth by controlling translation of proliferation-regulating genes

Lisa B Earnest-Noble et al. Nat Cancer. 2022 Dec.

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.

© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

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Figures

Figure 1 –

Figure 1 –. Isoleucine isoacceptors are differentially modulated in isogenic poorly and highly metastatic breast cancer pairs.

(a) Volcano plot representing log2 fold change vs. −log p value of POL3RA ChIP sequencing analysis of MDA-LM2 cells vs. MDA-MB-231 Parental cells. (b) tRNAIleUAU quantification by specific tRNAIleUAU probe RT-qPCR normalized to 18S probes of highly metastatic LM2 lines relative to their parental MDA-MB-231 and HCC1806 cell lines. (c) tRNAIleGAU quantification by specific tRNAIleGAU probe RT-qPCR normalized to 18S probes of highly metastatic LM2 lines relative to the parental MDA-MB-231 cell line. (d,e) Relative pre-tRNA abundance of tRNAIleUAU and tRNAIleGAU across multiple primers covering distinct genetic loci using RT-qPCR of MDA-LM2 vs. MDA-MB-231 (d) & HCC1806-LM2C vs. HCC1806 Parental cells (e). (f) Relative tRNAIleUAU/tRNAIleGAU ratios quantified by fluorescent intensity normalized to DAPI of breast tissue microarrays, stratified by normal tissue or breast cancer stage I & II, III, measured by FISH with LNA targeting tRNAIleUAU or tRNAIleGAU. Two-sided un-paired student’s t-tests performed, p-values p<0.05, p<0.01, p<0.001 represented as *, **, ***, respectively.

Figure 2 –

Figure 2 –. tRNAIleUAU promotes & tRNAIleGAU suppresses metastatic colonization.

(a-b) Bioluminescent imaging post tail vein injection of 1×105 of MDA Parental (a) or 1.5×105 HCC1806 (b) cells overexpressing tRNAIleUAU or control with representative lung histology stained with H&E; n=5 in each cohort. (c) Quantification of lung metastatic nodules post extraction after tail vein injection of 5×104 LM2 CRISPR cells guides targeting eGFP or tRNAIleUAU, with representative histology for control & tRNAIleUAU guide 1; n=5 in each cohort. (d) Bioluminescence imaging after tail vein injection of 1×105 of MDA LM2 cells overexpressing tRNAIleGAU or control with representative images; luminescence expressed as Radiance p/sec/cm2/sr; n=5 in each cohort. (e,f) Same as (d) with MDA Parental CRISPRi cells with guides targeting either control or tRNAIleGAU (e) or shRNA targeting control or tRNAIleGAU with representing H&E lung histology (f). (g) Quantification of lung metastatic nodules post extraction after tail vein injection of 5.0 × 104 MDA cells with CRISPRi targeting tRNAIleGAU and tRNAIleUAU overexpression or control; n=5–6 in each cohort, with representative histology for control & tRNAIleGAU depletion and tRNAIleUAU overexpression. (h) Bioluminescent imaging post tail vein injection of 1.5×105 HCC1806 cells with shRNA targeting tRNAIleGAU and tRNAIleUAU overexpression or control; n=5–6 in each cohort. (i) Quantification of lung metastatic nodules post extraction after tail vein injection described in (a) with representative histology for control & tRNAIleGAU depletion and tRNAIleUAU overexpression. (j) Quantification of lung metastatic nodules post extraction after tail vein injection of 1×106 of MDA MB 468 cells shRNA targeting tRNAIleGAU and tRNAIleUAU overexpression or control with representative images, n=5 in each cohort. (k) Primary tumor growth curves of NSG mice injected with either 1×106 MDA MB 231 control or tRNAIleGAU depletion tRNAIleUAU overexpression cells in mammary fat pad with 1:1 Matrigel, volume measured in cubic mm, n=6 mice. (l) Bioluminescent imaging of lungs and quantification of lung metastatic nodules post extraction from orthotopic mammary fat pad injections described in (k). Statistics utilized include 2-way ANOVA for imaging and two-sided unpaired student’s t-test for nodule quantification, p-values p<0.05, p<0.01, p<0.001 represented as *, **, ***, respectively.

Figure 3 -

Figure 3 -. Cell Cycle and Response to Stress gene expression and phenotypes characterize tRNAIle modulations.

(a,b) Reactome pathways significantly enriched in AUA (a) or AUC (b) by relative synonymous codon usage (RSCU) using iPAGE. (c,d) GO function terms for positive and significant TE changes from ribosomal profiling (c) or label free quantification by mass spectrometry (d) in tRNAIleGAU depletion and tRNAIleUAU overexpression cells versus control. (e) Quantification of Ki67 immunofluorescence staining in MDA MB 231 tRNAIleGAU depletion and tRNAIleUAU overexpression cells versus control. (f,g) Relative cell counts of MDA MB 231 control & tRNAIleGAU depletion tRNAIleUAU overexpression cells exposed to 0.5% hypoxia (f) or treated with 200uM H202 (g) for 3 days. (h) Venn Diagram of overlapping datasets to identify downstream effectors – includes high RSCU tRNAIleUAU score (top 50%), and genes with significantly positive changes in TE and proteomics in both MDA LM2 vs. MDA Parental cells and tRNAIleGAU depletion tRNAIleUAU overexpression cells vs. control. (i) Relative cell count of MDA tRNAIleGAU depletion tRNAIleUAU overexpression cells treated with control or SMNDC1 siRNA in 0.5% hypoxia for 2 days. Western performed on siRNA cells on day 3. (j) Relative cell counts of MDA tRNAIleGAU depletion tRNAIleUAU overexpression cells treated with control or LSM6 siRNA for 3 days. (k) Relative cell counts of MDA tRNAIleGAU depletion tRNAIleUAU overexpression cells transduced with shRNA targeting either control or PYCARD for 3 days. (l) Bioluminescent imaging of lungs of NSG mice injected with 1×106 MDA tRNAIleGAU depletion tRNAIleUAU overexpression cells with either siRNA targeting SMNDC1, LSM6, PYCARD, or control, n=3–5 mice per group. (m) Quantification and H&E histology of lung metastatic nodules post extraction after tail vein injections described in (l). (n,o) LICOR Western quantification of either Flag tagged wildtype (n) or all AUA to AUC codons (n) SMNDC1 expression relative to reporter control luciferase (all Ile AUU) 24 hours post transfection in either MDA control or tRNAIleGAU depletion tRNAIleUAU overexpression cells. Representative images below. Statistics utilized include 2-way ANOVA for imaging and two-sided un-paired student’s’s t-tests performed, p-values *, **, **** indicated as p<0.05, p<0.01, and p<0.0001, respectively.

Figure 4 –

Figure 4 –. Translational efficiency of AUA enriched transcripts is dependent on tRNAIleGAU abundance.

(a) Genes with a high abundance of AUA codons (using RSCU scores) were significantly enriched among genes upregulated in polysomes (corrected for their transcript changes) in tRNAIleGAU depleted tRNAIleUAU overexpression cells versus control MDA-MB-231 cells. The statistical significance of these enrichments was assessed using mutual-information calculations and associated Z score (based on randomized input vectors) and robustness scores (based on jackknifing tests). 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 (for details, see Goodarzi et al., 2009). (b) Same as (a) except analyzed for AUC codon enrichment, showing significantly depletion among genes upregulated in polysomes (corrected for their transcript changes) in tRNAIleGAU depleted tRNAIleUAU overexpression cells versus control MDA-MB-231 cells. (c) Ribosomal AUA codon dwelling times as estimated by CELP bias coefficients (higher bias coefficient indicates longer dwelling time). Univariate regression coefficients estimating the effects of tRNAIle modulated MDA cells. A 95% confidence interval excluding zero (not overlapping the vertical line x=0) means that the tested effect was significant at α=0.05 (p<0.05). (d) Hypergeometric distribution shown as a z- score of AUA codon enrichment of polysome transcripts represented as log2 fold change of tRNAIleGAU depleted tRNAIleUAU overexpression cells versus tRNAIleGAU depleted MDA MB 231 cells, stratified in bins of 10, increased log2 fold change from left to right. AUA codon representation visualized as a value ranging from −10 to 10 relative to the average. (e) Same as (d) except analyzed for AUC codon enrichment. (f) Ribosome abundance of tRNAIle quantified by specific tRNAIleGAU (left) and tRNAIleUAU (right) probes. RT-qPCR normalized to 5S probes of tRNAIle modulated MDA cells from polysome fractions, measured as % Input. (g) Ribosomal ratio of tRNAIleUAU/ tRNAIleGAU abundance quantified by specific tRNAIleGAU probe and tRNAIleUAU probe RT-qPCR normalized to 5S probes of tRNAIle modulated MDA cells from polysome fractions, measured as % Input. (h) Model depicting how tRNAIle abundance shifts alter translational dynamics and metastatic phenotypes. Two-sided un-paired student’s’s t-tests performed, p-values represented as *, ** as p<0.05, p<0.01 respectively.

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