Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

Abstract

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.

Figure 1.

Plasmid born in cellulo production of human tRNAs from the U6 promoter. (A) Schematic of the U6-tRNAbox cassette used for overexpression of human tRNAs. (B) The sequence of U6-tRNAbox cassette including U6 promotor features. Individual U6-tRNAbox cassettes with various tRNA sequences subcloned between ApaI and KpnI sites of the ‘tRNAbox’ were always cut at the specific restriction sites within the multicloning sites (MCS) A and B and inserted into the reporter of interest cleaved with the corresponding restriction sites. The PSE, TATAbox, the +1 position of the human U6 promoter, and a synthetic terminator are indicated in red.

Figure 2.

Overexpression of tryptophan and tyrosine tRNAs increase stop codon readthrough in human cells. (A) Schematic of the readthrough reporter containing the U6-tRNAbox cassette. For more details please the main text. (B) Readthrough measurements using the UAG—TMV reporter (see panel C) with or without selected human tRNAs in the HEK293T cells. The schematics of base-pairing between individual tRNAs and the UAG stop codon is shown to the right of the name of each construct, followed by the gene identity with the tRNA sequence and the relative percentage of the UAG readthrough. The tRNA sequences were individually placed under the U6 promoter of the PBB168 reporter or its sense control PBB172 reporter (see Figure 1). The tQ01s (marked by an asterisk) is the mutated variant of tQ01; a single-point mutation in the anticodon is underlined. (C) Readthrough measurements of all three stop codon reporters in Hek293T cells overexpressing Trp tRNA (tW01) or Tyr tRNA (tY01). The corresponding sequences of these two tRNAs shown in panel 2B were placed under the U6 promoter of PBB171 (for the UGA reporter); PBB168 (UAG); PBB170 (UAA), or its sense control PBB172. As an example, the DNA spacer sequence containing the UGA stop codon followed by six nucleotides representing the TMV stop codon nucleotide context is shown below. Changes in readthrough levels to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P<0.05). (D) The UGA – Trp tRNA (tW01) readthrough measurements as in panel C only two copies of the U6-tRNA^Trp cassettes was added (plasmid PBB288). Changes in readthrough levels to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05). (E) The UAG—Tyr tRNA (tY01) readthrough measurements as in panel D (plasmid PBB292). Changes in readthrough levels to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05).

Figure 3.

Depletion of tryptophan and tyrosine aminoacyl-tRNA synthetases decreases stop codon readthrough. (A) The UAG and UGA readthrough measurements in HEK293T cells after siRNA-mediated downregulation of eRF1 (upper panel), YARS (middle panel), and WARS (lower panel). For details please see Materials and Methods. Changes in readthrough levels to non-targeted siRNA control were analyzed by the Student's t-test (mean + SD; n = 2 or n = 6) and shown to be statistically significant for those values marked (P< 0.05). (B) mRNA and protein levels of selected factors in individual knock-downs determined at the time of the readthrough measurement; same seeding as in panel A was used. The mRNA levels were established by RT-qPCR and quantified as described in Materials and Methods.

Figure 4.

Tryptophan tRNA overexpression increases SC-RT on reporters bearing stop codon contexts of mRNAs encoding human proteins known to be natural readthrough substrates. The UGA readthrough measurements in Hek293T cells overexpressing tRNA^Trp (tW01) or tRNA^Tyr (tY01) transfected with the pSGDluc-based plasmids bearing genomic sequences surrounding stop codons of three genes of interest as spacers, or UGAC as a basal readthrough control. Their respective sense (UGG) controls were used in parallel. Changes in readthrough levels to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05). For details please see Materials and Methods.

Figure 5.

Tryptophan tRNA overexpression increases SC-RT on reporters bearing SCCs of mRNAs encoding viral proteins from two human pathogenes - Alphavirus and Coltivirus. (A) Spacer sequences of the p2luci reporters containing the UGA stop codon genomic sequences of Sindbis virus, CTFV virus, or the UGA-CUAG human readthrough permissive nucleotide context. (B) The UGA readthrough measurements in Hek293T cells overexpressing tRNA^Trp (tW01) transfected with the p2luci reporters described in panel A; their respective sense (UGG) controls were used in parallel. Changes in readthrough levels to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05). For details, see Materials and Methods.

Figure 6.

Tryptophan tRNA overexpression enhances restoration of a functional p53 protein production from a mutant PTC-containing mRNA. (A) The schematics of the p53-based functional assay for determination of production of functional p53 protein from PTC-containing mRNAs. The full-length p53 as well as its three PTC-terminated forms are co-expressed from the CMV promoter together with the luciferase under the control of the p53-dependent promoter (from Bcl-G gene) containing p53-binding sites (BS) surrounding its TATA box. In addition, the luciferase-based reporter also contains and expresses the Trp or Tyr U6-tRNAbox cassettes described in Figure 1B. (B) The p53-dependent luciferase activity from PTC-containing p53 mRNAs normalized to the luciferase activity obtained with the full-length p53 protein. p53 null cells H1299 were co-transfected with p53 expression plasmids and luciferase/tRNA reporters described in panel A, as well as with a plasmid bearing β-Galactosidase for internal normalization and subjected to measurement analyses as described previously (46). Changes in levels of p53 dependent luciferase activity to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05). (C) Restoration of the p53 functional protein expression from the PTC-containing mRNA (W53UGA-U) responds to the Trp tRNA (tW01) overexpression. Same as in panel B, except that luciferase reporters PBB321, PBB301 and PBB330 bearing none, one or two tRNA^Trp cassettes were used. Changes in levels of p53 dependent luciferase activity to no tRNA control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05). (D) Overnight G418 treatment (200 μg/ml) boosts restoration of the p53 functional protein expression from the PTC-containing mRNAs (W53UGA-U and W146UGA-G). Changes in levels of p53 dependent luciferase activity to no drug control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P< 0.05). Production of the full length p53 protein was monitored by the ‘Western-like’ analysis using the Jess system from Protein Simple; for details please see Materials and Methods. (E) Combining G418 treatment with overexpression of Trp-tRNA displays an additive effect on restoration of the p53 functional protein expression from the PTC-containing mRNA (W53UGA). Changes in levels of p53 dependent luciferase activity to no tRNA no drug control were analyzed by the Student's t-test (mean + SD; n = 2) and shown to be statistically significant for those values marked with the asterisk (P<0.05).