Overexpression of initiator methionine tRNA leads to global reprogramming of tRNA expression and increased proliferation in human epithelial cells

Abstract

Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNAi(Met) in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNAi(Met) significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.

FIGURE 1.

Experimental strategy for tRNA overexpression. The tRNA gene of interest with 200-bp flanking regions was cloned into a mammalian expression vector, then stably transfected into the human cell line. The stable cell line was then characterized in terms of tRNA expression profile, metabolic activity, and cell proliferation.

FIGURE 2.

tRNA_i^Met overexpression generates unique tRNA expression profiles. (A) Individual tRNA abundances in 184A1–tRNA_i^Met and 184A1–tRNA_e^Met cell lines. Individual tRNA abundance values are shown for 184A1–tRNA_i^Met (black) and 184A1-tRNA_e^Met (gray) relative to an empty vector control cell line (set to 1, red line). A value of 1 indicates no change, a value <1 indicates a decrease, and a value >1 indicates an increase in tRNA levels relative to the control cell line. Data are grouped according to amino acid type. The tRNA_i^Met and tRNA_e^Met probes are labeled Met-i and Met-CAU, respectively. Where error bars are present, values are averages from dye-swapped experiments and error bars indicate standard deviation. One sample t-test was performed to determine the statistical significance of the changes: *P-value <0.05. (B) Validation of microarray data by dot blot. As in A, relative tRNA abundance is defined as the ratio between the indicated cell line and the control cell line. Relative tRNA abundance values obtained by microarray (white) and dot blot (black, average of three replicates, error bars indicate standard deviation) are plotted for tRNA_i^Met and tRNA_e^Met in the three 184A1 cell lines generated for this study (control, 184A1–tRNA_i^Met and 184A1–tRNA_e^Met). (C) Median tRNA abundance upon tRNA_i^Met overexpression. Median values for 184A1–tRNA_i^Met and MCF10A–tRNA_i^Met relative to control cell lines (set to 1, red line). Median values for nuclear-encoded tRNAs (gray) and mitochondrial-encoded (white) tRNAs are shown. The upper and lower bars indicate the range of individual tRNA abundances. (D) Heat map of tRNA abundances upon tRNA_i^Met overexpression. Relative tRNA abundance levels of nuclear and mitochondrial-encoded tRNAs in 184A1–tRNA_i^Met and MCF10A–tRNA_i^Met are shown as TreeView images. Data are grouped according to amino acid type. Green indicates a decreased level of expression, red indicates an increased level of expression, and black indicates no change in expression level relative to the reference sample. (E) Individual tRNA abundances in 184A1–tRNA_i^Met compared with MCF10A–tRNA_i^Met. Individual tRNA abundance values for 184A1–tRNA_i^Met and MCF10A–tRNA_i^Met are relative to the corresponding control cell lines. (F) Individual nuclear-encoded tRNA abundances in two breast cancer lines, MDA-MB-231 (left) and BT-474 (right) compared with 184A1–tRNA_i^Met and MCF10A–tRNA_i^Met. Individual tRNA levels for the two breast cancer cell lines, MDA-MB-231 and BT-474, are relative to the breast epithelial MCF10A cell line. Individual tRNA levels for the 184A1–tRNA_i^Met and MCF10A–tRNA_i^Met cell lines are relative to the corresponding control cell lines.

FIGURE 3.

tRNA_i^Met overexpression leads to increased cell metabolism and proliferation. Data are shown for the 184A1 cell lines 184A1–control, 184A1–tRNA_i^Met, and 184A1–tRNA_e^Met. T-tests were performed to determine the statistical significance of the differences observed relative to 184A1–control: *P-value <0.05. (A) Metabolic activity. Mitochondrial metabolic activity was measured by WST1, which relies on the activity of mitochondrial dehydrogenases. Cytosolic metabolic activity was measured by Calcein AM, which relies on the activity of cytoplasmic esterases. Where error bars are indicated, assays were performed in triplicate and the error bars indicate standard deviation. (B) Cell proliferation. Cell proliferation was measured over 5 d by Hoechst DNA staining. The assays were performed in triplicate; error bars indicate standard deviation.