Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes

Abstract

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species.

Fig 1. Crossing worms that contain a read-through reporter with tRNA suppressor mutants was used to report tRNA expression.

(A) A schematic representation of the tRNA reporter system. To monitor tRNA expression, we created worms that are homozygous for the sup mutation, the smg-2 mutation, and the reporter construct. (B) mCherry expression is a proxy for tRNA levels, and GFP reports the activity of the rps-0 promoter. Mixed-stage populations of control worms (no read-through activity) and sup-7 mutants are shown.

Fig 2. Copies of tryptophan tRNA genes display different expression patterns.

(A) Representative images of young adult worms; all eight tryptophan tRNA reporter strains are displayed (90/90 worms exhibited the same expression patterns). mCherry expression is a proxy for the expression of specific tryptophan tRNA genes. Images were taken under exposure conditions that also allow detecting expression in tissues where the tRNA levels are relatively low. (B) Quantification of total worm mCherry fluorescence. All strains were analyzed when the worms were the same age (young adults), using the same exposure parameters. Shown are averages of means, ± SEM, normalized to the expression levels detected in the sup-7 strain. Three experiments were conducted, and 30 worms of each strain were scored in each experiment. *** p-value<0.001 (C) The results that were obtained using the fluorescent reporter were plotted against the Chip-seq measurements of pol III occupancy [52].

Fig 3. Differential expression of tryptophan tRNAs in C. elegans throughout time.

(A) Representative images of three tryptophan tRNA reporter strains at various stages and ages. Images were taken under exposure conditions allowing optimal visualization of the expression patterns. (B) Quantification of total worm mCherry fluorescence was normalized to total worm GFP fluorescence, in the tRNA reporter strains, at different stages and ages. Measurements of the fluorescence in the different strains were taken at the same time using the same exposure parameters. Shown are averages of means, ± SEM; three experiments were conducted, N = 30 worms from each strain in each stage per experiment.

Fig 4. Sup-7 tRNA expression predicts the worms' lifespan.

(A) The intensity of mCherry fluorescence (normalized to GFP) in sup-7 worms at day 6 (X-axis) correlates with the remaining lifespan of the same worm (Y-axis, two tailed p-value for correlation significance). (B) Shown are representative images of day 7 worms that were fed with bacteria that induce daf-2 RNAi (right panel) or with bacteria that express an empty RNAi vector (control, left panel).

Fig 5. The promoter of the host gene is required for proper tRNA expression.

(A) Schematic representation of the sup-7 constructs. All the constructs that are shown here include the sup-7 suppressor tRNA. (1) A sup-7 mutation in the genome (sup-7). (2) A construct that includes the C03B1.2 gene, including its native promoter (“sup-7 (+) promoter”). (3) A construct that includes only the C03B1.2 gene, without a promoter (“sup-7 (-) promoter”). (4) A construct in which the endogenous promoter of the C03B1.2 gene was swapped with the hsp-16.48 promoter (“sup-7 (+) HSP promoter”). (5) A construct in which the endogenous promoter of the C03B1.2 gene was swapped with a myo-3 promoter (“sup-7 (+) myo-3 promoter”). (B) The expression pattern of a tRNA in L1 transgenic worms that express an extrachromosomal "sup-7 (+) promoter" construct (middle panel) is highly similar to the expression pattern displayed by mutants that harbor the genomic suppression mutation (upper panel). On the other hand, L1 transgenic worms expressing extrachromosomally the "sup-7 (-) promoter" construct exhibit aberrant expression (lower panel) and sterility (Table 2), and therefore, are significantly different from the sup-7 (control) strain. No expression was found when construct 6 was injected (Table 2). (C) Quantification of the total mCherry fluorescence levels normalized to sup-7 control worms (n = 15; error bars are s.d., * p < 0.01). (D) At 15°C only worms that do not have a sup-7 mutation (the RRS strain) and the worms that express the “sup-7 (+) HSP promoter” transgene are fertile. Worms harboring a genomic mutation in sup-7 (control, sup-7) as well as transgenic worms that express the “sup-7 (+) promoter” transgene are sterile. At 22°C all strains are fertile (an average of 3 experiments ± SEM, the brood size of 5 worms was monitored in each experiment). (E) Transgenic worms expressing the “sup-7 (+) HSP promoter” transgene are healthy at 15°C and sick at 25°C. Sickness is manifested as a 10-fold reduction in the number of transgenic progeny and the appearance of severe deformations (an average of 3 experiments ±SEM, N = 100 per experiment). rol-6 was used as a co-injection marker. (F) A deformed worm expressing the “sup-7 (+) HSP promoter” transgene at 25°C. Shown are mCherry (red), GFP (green), and phase (gray) images. (G) The few transgenic worms that survives and expressed the “sup-7 (+) myo-3 promoter” transgene displayed expression of sup-7 in muscles and were sterile (Table 2).

Fig 6. Conservation of the pairing between tRNAs and hosting or neighboring protein-coding genes among nematodes.

Shown are the fractions of conserved genomic architectures in different nematodes in comparison to C. elegans. Each bar denotes the conservation of localization of a given tRNA type (anticodon) with respect to either the tRNA-hosting gene or the adjacent protein-coding genes. Shown are the fractions of conservation of a given anticodon within the transcripts of a specific hosting protein-coding gene (red bars), and the conservation of the adjacent upstream (yellow bars) or downstream (blue bars) protein-coding genes in the vicinity of a given tRNA type (anticodon). The yellow and blue bars were generated based on all the individual tRNA genes that were not localized within the transcripts of protein-coding genes.

Fig 7. The difference in the PolIII occupancy of intronic and non intronic tRNA genes in C. elegans.

Shown are histograms of PolIII occupancy of young adult worms in intronic (red bars) and non-intronic (blue bars) tRNAs. Occupancy is given in terms of Q-values (-log10 scale). Red bars correspond to intronic tRNAs; blue bars represent non-intronic tRNAs (Wilcoxon rank sum test calculated p-value = 1.31*10⁻⁰⁴).

Fig 8. A schematic model for regulating intronic tRNAs by the host gene promoter.

tRNA genes are independent entities transcribed by RNA pol III. We showed here that different copies of tRNA are transcribed in a tissue specific manner. One level of regulation can arise from tRNA genes that reside inside intron of a protein coding gene. Shown here are the possible interaction between Pol II gene promoters and Pol III-mediated transcription of intronic tRNAs. Using heat shock promoters we show that promoter repression in cold temperatures can silence the intronic tRNA, and that release of repression in high temperatures enables tRNA expression. The possibility that certain tRNAs can affect the host gene’s expression is also considered (top). Once the gene’s promoter is silenced, tRNA transcription is suppressed (bottom).