Diversity of tRNA genes in eukaryotes

Abstract

We compare the diversity of chromosomal-encoded transfer RNA (tRNA) genes from 11 eukaryotes as identified by tRNAScan-SE of their respective genomes. They include the budding and fission yeast, worm, fruit fly, fugu, chicken, dog, rat, mouse, chimp and human. The number of tRNA genes are between 170 and 570 and the number of tRNA isoacceptors range from 41 to 55. Unexpectedly, the number of tRNA genes having the same anticodon but different sequences elsewhere in the tRNA body (defined here as tRNA isodecoder genes) varies significantly (10-246). tRNA isodecoder genes allow up to 274 different tRNA species to be produced from 446 genes in humans, but only up to 51 from 275 genes in the budding yeast. The fraction of tRNA isodecoder genes among all tRNA genes increases across the phylogenetic spectrum. A large number of sequence differences in human tRNA isodecoder genes occurs in the internal promoter regions for RNA polymerase III. We also describe a systematic, ligation-based method to detect and quantify tRNA isodecoder molecules in human samples, and show differential expression of three tRNA isodecoders in six human tissues. The large number of tRNA isodecoder genes in eukaryotes suggests that tRNA function may be more diverse than previously appreciated.

Figure 1

tRNA genes and isodecoder genes in 11 eukaryotes. (A) Cladogram of the organisms based on the NCBI taxonomy browser (40,41) which include two single cell yeast, worm, fruit fly, fugu, chicken and five mammals, dog, rat, mouse, chimp and human. The fraction of tRNA isodecoder genes among all tRNA genes is indicated. *: ∼ 400 genes in the tRNA^Lys(CTT) isoacceptor class in the dog genome are not included in this count. (B) The number of tRNA genes (left), isoacceptors (middle) and isodecoders (right) in these organisms.

Figure 2

Gene copy numbers of tRNA isoacceptors versus the number of occurrence or the number of isodecoders. (A) Plot of the gene copy number of tRNA isoacceptors and the number of occurrence for each isoacceptor class. (B) Plot of the gene copy number of tRNA isoacceptors and the number of tRNA isodecoders for each isoacceptor class. A good linear correlation (R-value between 0.89 and 0.92) exists between the gene copy number of tRNA isoacceptors and the number of tRNA isodecoders in the three mammals.

Figure 3

Comparative sequence analysis of the tRNA^Ser(AGA) isoacceptor family across six species. S.c.: budding yeast; C.e.: worm; D.m.: fruit fly; M.m.: mouse; P.t.: chimpanzee; H.s.: human. ‘-Nx’ indicates the gene copy number. For the non-mammalian species, the tRNA sequence variants are most similar within the same organism. The tRNA sequence variants are conserved across mammalian species. Each phylogenetic branch has unique sequence signatures (e.g. for the 2–71 bp, yeast sequence is GU; worm and fruit fly sequence is CG; and mammal sequence is TA).

Figure 4

Frequency of human isodecoder gene variations. Percentages indicate observed changes in each region divided by the total number of nucleotides assessed in that region. (A) Percent sequence variations in the A and B boxes which correspond to internal promoter regions of RNA polymerase III. (B) Percent sequence variations in nine regions according to the tRNA secondary structure. Invariant and anticodon nucleotides in all tRNAs are shown as filled black or gray circles. Percentages in parentheses (stems only) indicate sequence changes that result in non-Watson–Crick and non-GU base pairs.

Figure 5

Detection of single nucleotide change in a model 30mer RNA by ligation. (A) The basic strategy. RNA oligonucleotides with a single nucleotide difference are used as templates for the ligation of two complementary oligonucleotides by T4 DNA ligase. To find the optimal ‘solution’ for each sequence at the 15th position (open and filled black circles), 28 oligonucleotides containing different sequences and backbone modifications at the ligation junction are tested. These oligos are named ‘floaters’ to distinguish them from the other oligonucleotide substrate that are the same in each set of the ligation reactions (‘anchors’). The ligation junction is located 3′ to the 15th position (set I), 5′ to the 15th position (set II) and 3 nt away from the 15th position (set III). (B) Ligation reaction using a defined mixture of C15 and G15 30mer RNAs (left). The particular floater used (II-mG) is selected as the best solution that prefers C15 over G15 after screening 24 floaters in separate experiments. (Right) Quantification of the ligation products using the D-oligos (II-mG and II-dC floaters) and the N-oligo (III-dC+3) for C15 versus G15.

Figure 6

Detection of tRNA isodecoder distribution in human samples. (A) Simultaneous detection of three tRNA isodecoder pairs plus yeast tRNA^Phe standard in a total tRNA mixture from HeLa. Asterisk (*) indicates a ligation product derived from the mixture of N-oligos without tRNA (also present weakly in the ‘No RNA’ lane). (B) Simultaneous detection of three tRNA isodecoder pairs in a total tRNA mixture from six human tissues. (C) Relative amount of tRNA isodecoder after normalization to the total amount of its corresponding tRNA isodecoder pair in each tissue as compared to brain. (D) ‘Absolute’ ratio of tRNA^Pro(CGG)-U39 in each tissue obtained from the ligation reactions using both D-oligos for tRNA^Pro(CGG)-U39 and tRNA^Pro(CGG)-C39.