Visualizing tRNA-dependent mistranslation in human cells

Abstract

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10 in yeast) with a limited impact on phenotype. Previously, we selected a tRNA containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNA (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 2-5% in HEK 293 cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.

Mistranslating tRNA identifies a deleterious S213P mutation in theSaccharomyces cerevisiae eco1-1allele

“…In contrast to the concerns addressed by Crick in his "Frozen Accident Theory" (Crick 1968), mistranslation is not catastrophic for cell viability. At levels in the 3-5% range mistranslation has minimal affect on yeast cell viability Lant et al 2017;Berg et al 2019). For some key cellular proteins, 3-5% mistranslation is sufficient to restore viability.…”

“…Loss of fidelity at either step can lead to mistranslation. Mistranslation is dramatically increased by tRNA variants that are inaccurately aminoacylated or contain mutations within their anticodon (Geslain et al 2010;Hoffman et al 2017;Lant et al 2017;Berg et al 2017;Zimmerman et al 2018). Nucleotides in the tRNA that are recognized by a specific aaRS are called identity elements and consist of single nucleotides, nucleotide pairs, and structural motifs (de Duve 1988;Hou and Schimmel 1988;Giegé et al 1998).…”

Targeted sequencing reveals expanded genetic diversity of human transfer RNAs

“…Mistranslation is a general feature of the translation process which has a basal error rate of approximately one misincorporated amino acid in every 10 4 to 10 5 codons [55,56]. Many tRNA variants dramatically increase mistranslation in bacterial, fungal, and mammalian cell culture systems [37,39,[57][58][59][60]. For example, we identified a variant of yeast tRNA Pro that results in~5% proteome wide alanine for proline mistranslation with a minimal effect on cell growth in yeast [39].…”

“…Unlike other isoacceptors, the anticodon of alanine, serine, leucine and selenocysteine tRNAs has no role in aminoacylation. If mutated and expressed, these tRNA variants could result in mistranslation [37]. We looked for variants with the potential to mistranslate either through mutations to the anticodon or by possessing a G3:U70 base-pair, the major identity element for AlaRS [37][38][39][40].…”

Transfer RNA function and evolution

“…Surprisingly, a perfect proteome is not a pre-requisite for cellular viability even in the context of human cells. Lant et al demonstrated that a single tRNA mutant can lead to significant mistranslation in human cells [17]. This was accomplished by expressing an Ala accepting tRNA Pro G3:U70 variant in HEK 293 cells.…”