Tissue-specific differences in human transfer RNA expression

Abstract

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.

Figure 1. The tRNA Microarray

(A) Schematics of fluorescence labeling of tRNA in a total RNA mixture. Ribonucleotides in the labeling oligos are shown in blue, and deoxyribonucleotides are shown in black. 5-NU, 5-allylamino-uridine; p, 5′ phosphate. All tissue samples are labeled with Cy3 and Cy5. At least two arrays are run for every sample pair. Array 1 used Cy3-labeled sample No. 1 and Cy5-labeled sample No. 2, and array 2 used Cy5-labeled sample No. 1 and Cy3-labeled sample No. 2. (B) Microarray images of one of the 32 blocks containing 100 spots hybridized with samples from HeLa (H), mouse kidney (M), and C. elegans whole animal (C). In the schematics below, black squares indicate the position of probes for the human (left), mouse (middle), and C. elegans (right) tRNAs. (C) Fluorescence intensity of a HeLa sample hybridized to the nuclear encoded tRNA probes (left) or mitochondrial encoded tRNA probes (right). “Human” indicates signals for the designated human probes; “other,” signals for Drosophila and C. elegans probes; and “mouse,” signals for mouse mitochondrial tRNA probes. (D) Dynamic range of Cy5/Cy3 ratio changes as a function of Cy3 intensity.

Figure 2. Overview of Relative tRNA Abundance among Eight Human Tissues

Red line indicates the same level as brain. (A) Relative ratios of each human tRNA probe for ovary or spleen versus brain sorted according to the ratios for ovary. Left, nuclear encoded tRNAs; right, mitochondrial encoded tRNAs. (B) Mean and median values of the nuclear tRNA probes for seven tissues versus brain. (C) Mean and median values of the mitochondrial tRNA probes.

Figure 3. Comparative Expression of Nuclear and Mitochondrial Encoded tRNAs among Eight Human Tissues and Two Cell Lines Shown as TreeView Images [37]

All tissue data are normalized to the median ratio in Figure 2B and 2C. The mean and median values for the HeLa/HEK293 cell lines are 1.19 ± 0.22 and 1.20 for nuclear tRNA probes and 1.48 ± 0.37 and 1.42 for mitochondrial tRNA probes. Green indicates decreased level of expression; red, increased level of expression in the indicated tissue over brain; gray, not determined due to very low signal intensity. Data are grouped according to codon-reading abilities (isoacceptors) (A) and corresponding amino acid types (B).

Figure 4. Linear Correlation of Relative tRNA Abundance to Codon Usage of Tissue-Specific Genes that Are Expressed at High Levels

The r- and p-values are indicated in each graph. (A) Liver/brain, all data points. Excluding the two outlying data points (circled) gives a linear fit with r = 0.78, p < 0.0001, and a slope of 1.0 ± 0.2. Inclusion of these two data points gives a linear fit with r = 0.62, p = 0.00098, and a slope of 0.8 ± 0.2. (B) Three individual isoacceptors across all five tissues show linear correlation with r = 0.90 to 0.94 and p = 0.016 to 0.039. (C) Two sets of four tRNA^Arg isoacceptors in liver and thymus show linear correlation with r = 0.93 and 0.97 and p = 0.067 and 0.033, respectively.