Microarray screen identifies miRNAs linked to UPR induction. Up- and down-regulated miRNAs populations in cells transfected with the mutagenic tRNAs. Number of miRNAs activated or repressed in cells transfected with tRNA^Ser(Lys), tRNA^Ser(His), tRNA^Ser(Ile), compared with cells transfected with wild-type tRNA^Ser. Graded activation and repression of miRNAs can be observed.

Chimeric tRNA as adaptors to mistranslate specific codons. (a) Schematic figure of chimeric tRNA concept: chimeric tRNAs are engineered by substituting the wild-type anticodon in tRNA^Ser to specific triplets coding for other amino acids. The chimeric tRNA^Ser will only be charged with serine by seryl-tRNA-synthetase, no matter what the anticodon sequence is. Therefore, expression of chimeric tRNA^Ser will produce mistranslated proteins. (b) GFP fluorescence was used as a marker to measure the impact of chimeric tRNA expression on the cell. Fluorescence was analysed by flow cytometry. A histogram depicting the different effect of each chimeric tRNA is shown. Mean values + standard deviations from three independent transfections are given.

Activation of UPR by chimeric tRNAs. (a) Kinetics of BIP and CHOP with persistent ER stress in vitro. Semi-quantitative RT–PCR (SQ-RT–PCR) was performed to check BIP and CHOP mRNA levels at the indicated different time points. (b) Activation of Xbp-1 and ATF6, and phosphorylation of eIF2α in cells expressing chimeric tRNAs. The presence of full-length (u) and spliced (s) Xbp-1 forms was checked by PCR. (c) BiP transcription induction in vivo. The expression levels of BiP (black staining) induced by each chimeric tRNA in a chicken embryo are proportional to the decrease in wt GFP fluorescence caused by the same tRNAs. Images are representative of three independent experiments 6 h post-electroporation (PE). GFP signal indicates the co-electroporated cells.

Statistical analysis. (a) Univariate analysis between the effect of each of the 10 tRNAs tested (in fluorescence values) and BLOSUM62 matrix substitution values, genome-wide codon frequency and, ER-, mitochondria-, nucleus- and cytoplasm-codon frequencies in related genes. The P-values for statistical significance are shown. BLOSUM62 matrix values were significantly associated with fluorescence. A significant association was also found for the ER- and mitochondria-related genes. No significant association was found for the cytoplasm and nucleus protein families. (b) Multivariate analysis graph. Fluorescence versus BLOSUM62 score and ER codon frequencies (P = 0.012) are represented. Fluorescence values determined for each of the ten mutagenic tRNAs are plotted against the frequency of the codons recognized by the same tRNAs in ER-related genes. For each tRNA the results of five independent transfections are shown. In this analysis, only the ER codon population statistically correlates with the profile of differential effects. The P-values calculated for the comparison with genome-wide, cytoplasmic, mitochondrial and nuclear codon frequencies are shown.

General physiological effects of chimeric tRNAs in vitro and in vivo. (a) Semi-quantitative RT–PCR of wt GFP mRNA shows that transcription is not affected by the expression of chimeric tRNAs. RPL19 mRNA was used as a control. Immunoblotting with an α-GFP antibody shows that GFP synthesis is reduced proportionally to the reduction observed in cell fluorescence (Figure 1b). β-Tubulin was used as a control. Error bars represent standard deviations from five independent experiments. (b) Total protein synthesis analysed by pulse-label experiments shows a reduction in total protein production after expression of chimeric tRNAs proportionally to the reduction in cell fluorescence (Figure 1b). Controls from cells not transfected and cells transfected with wild-type tRNA^Ser are shown. (c) Effect upon cell division of the chimeric tRNAs. Cells were counted at different times using flow cytometry. The data shows a reduction in growth rate for each tRNA that is to the reduction in cell fluorescence caused by the same molecule. (d) Effect in vivo of tRNA^Ser(Ile) upon cell division. BrdU incorporation was measured in vivo in chicken embryos electroporated with chimeric tRNA^Ser(Ile). A significant decrease in BrdU incorporation was observed in transfected embryos, indicating that the expression of chimeric tRNAs blocks cell proliferation during neural tube development. (e) Cell death and apoptosis measured in cultures transfected with chimeric tRNAs. Apoptosis was monitored by staining with propidium iodide, and the proportion of dead cells in each culture was measured by flow cytometry. The induction of cell death and apoptosis by each of the chimeric tRNAs tested was proportional to the reduction observed in cell fluorescence for the same tRNAs. (f) Induction of apoptosis in a chick neural cells expressing tRNA^Ser(Ile) was monitored by caspase-3 immunostaining. Sections from electroporated embryos were stained with an α-caspase-3 antibody (6, 12 and 18 h after electroporation) and the number of labelled cells was quantified. A significant increase of caspase-3-positive cells is detected. The white arrow indicates the electroporated side of the embryo. GFP shows transfected cells. Data are means + SD (graph).