Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF

Figures

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  FIGURE 1. (a) Cartoon of the synonymous recoding strategy for our constructs. All synonymous versions of the shble ORF are located in a bicistronic mRNA in tandem with the egfp ORF downstream, and under the transcriptional control of the cytomegalovirus (CMV)‐ promoter. The shble ORF is preceded by an invariant stretch of 18 nucleotides encoding for an AU1 epitope. Synonymous shble variants explore a sequence space associated to differences in GC3, CpG and TpA composition, as well as in match to the average human codon usage preferences and mRNA folding energy. (b) First two axes of a principal component analysis (PCA) of the compositional variables for the 13 shble versions used. The percentage of the total variance captured by each axis is given in parenthesis. GC3, percentage of G or C at the third codon nucleotide codons; freq_CpG, CpG dinucleotide frequency; freq_TpA. TpA dinucleotide frequency; folding, energy of the more stable structure predicted for the mRNA shble ORF estimated with UNAfold online tool (http://unafold.org) (Markham & Zuker, 2008); COUSIN_59, value of the COdon Usage Similarity Index of the shble version with respect to the average human codon usage (Bourret et…

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  FIGURE 2. Compositional variation of synonymous shble versions does not affect mRNA levels. (a) Box‐and‐whiskers plot showing relative levels of total heterologous mRNAs measured by RT‐qPCR from four or eight biological replicates. Within a given replicate, relative mRNA level values were normalized by the median value of all samples, allowing comparison across replicates. The positive control “empty” condition transcribed a monocistronic egfp mRNA while the 13 shble conditions (sh1 to sh13) transcribed a bicistronic shble_egfp mRNA. Letters present the results of a pairwise Wilcoxon rank sum test with B–H adjusted p‐values. Median values for samples labeled with the same letter are not statistically different (α = 0.05). (b) Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between the projection on the first axis of PCA in Figure 1 for each shble version, and the relative mRNA levels.

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  FIGURE 3. (a) SHBLE protein levels as a function of mRNA levels. Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between relative shble_egfp mRNA levels and relative GFP protein levels for the 13 shble synonymous versions. (b) SHBLE translation efficiency as a function of shble sequence characteristics. Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between the projection on the first axis of PCA in Figure 1 for each shble version, and the protein‐over‐mRNA ratio for data presented in panel (a). For all, values from a same biological replicate are represented by triangle, rectangle or circle shapes.

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  FIGURE 4. (a) GFP production from synonymous versions of a bicistronic shble_egfp mRNA. Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between proteomic‐based, sample‐normalized GFP intensity levels and the sum of fluorescence intensity of the cellular population for the 13 shble versions. Sums of fluorescence were calculated by integrating the fluorescence signal of 30,000 randomly selected cells in each transfection event. (b) Dot‐plot showing relative levels of heterologous proteins measured by label‐free proteomic from three biological replicates. For each sample, iBAQ values of SHBLE and GFP were normalized by the total iBAQ value of the sample. The positive control “GFP_mono” condition translated GFP from a monocistronic egfp mRNA while the 13 shble conditions translated SHBLE and GFP from a bicistronic shble_egfp mRNA. GFP values do not follow a normal distribution after a Shapiro normality test (p = 0.0014), hence the p‐values present the results of a pairwise Wilcoxon signed rank exact test. (c) Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between relative shble_egfp mRNA levels and proteomic‐based…

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  FIGURE 5. Relative SHBLE and GFP production from bicistronic shble_egfp mRNA. (a) Box‐and‐whiskers plot of the SHBLE/GFP proteins ratio for the 13 shble versions, stratified by their match to the average CUPrefs of the human genome. Letters present the results of a pairwise Wilcoxon rank sum test with B–H adjusted p‐values; α = 0.05; median values for samples labeled with the same letter are not statistically different. (b) Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between the projection on the first axis of PCA in Figure 1 for each shble version, and the SHBLE‐over‐GFP proteins levels. (c) Pearson's linear regression (black line) and 95% confidence interval of the fit (gray) between SHBLE translation efficiency and GFP translation efficiency. For panels (b) and (c), values from a same biological replicate are represented by triangle, rectangle or circle shapes.

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  FIGURE 6. Effect of shble_egfp bicistronic mRNA splicing on SHBLE and GFP protein levels. (a) Schematic representation of unspliced and spliced bicistronic mRNAs generated for shble versions shble#4, #6, #7, #10, and #13 and their respective original (splice‐able) or mutated (splice‐ablated) sequences. Figure should be read as follows, using shble#4 as an example: “sh4sp” refers to the sequence that undergoes splicing, while “sh4” refers to the sequence that has been mutated to ablate splicing. Splice donor (SD) and splice acceptor (SA) site positions relative to the shble AUG are indicated by discontinuous lines. (b) Box‐and‐whiskers plot showing the fraction of unspliced mRNA generated by splice‐able (e.g., “sh4sp”) and splice‐ablated (e.g., “sh4”) constructs, determined by Bioanalyzer. (c) Dot plot showing relative levels of heterologous proteins measured by label free proteomic from three biological replicates. For each sample, iBAQ values of SHBLE and GFP were normalized by the total iBAQ value of the sample. The positive control “GFP_mono” condition translated GFP from a monocistronic egfp mRNA while the 10 shble conditions translated SHBLE and GFP from a spliced or non‐spliced…