Page 108 - 2021_03-Haematologica-web

I. Boussaid et al.
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Figure 7. The codon adaptation index, coding sequence length and thermodynamic characteristics of the untranslated regions (UTR) in an mRNA are key determi- nants of its translation during normal erythropoiesis. (A-C) Bi-parametric scatter plot in a log scale of the codon adaptation index (CAI) and length (coding sequence [CDS]+UTR) of all human transcripts denoted by gray dots. (A) The top five percentile transcripts which are the more or the less translated after hemin induction of differentiation in K562 model (Mills et al.,35 GSE85864) are overlaid in red or blue, respectively. dH: day post hemin. (B) The top 15 transcripts most translated mRNAs in reticulocyte (Mills et al.,35 GSE85864) overlaid in green (C) The top 15 most expressed proteins in red blood cells (Gautier et al.,15 PXD009258) overlaid in green. (D) Dynamic repartition of proteins during erythropoiesis. Heatmaps show repartition of the protein copy numbers ranked according to their transcripts fea- tures (CAI, 5’ and 3’UTR structure and total length) in the rows and stages of differentiation in the columns. Each row represents 1/10 of the total scale of the cor- responding feature. For example, at the Epp stage 94.5% of the protein cellular content is issued from transcripts with a very high CAI (Top 10 % of CAI). Ret: reticu- locytes; Eep: erythrocyte-enriched population; Epp: erythrocyte-purified erythrocytes.
Validation of codon bias as a determinant of translation selectivity
To further validate the contribution of codon bias to translation regulation, we used relative synonymous codon usage (RSCU) as a second metric for codon bias measurement. This analysis confirmed that transcripts with a decreased ΔTE were enriched in optimal codons (Online Supplementary Figure S4A). We then performed luciferase assays using a dual luciferase vector harboring a firefly luciferase (Fluc) with a high CAI and a renilla
luciferase (Rluc) with a medium CAI. We also engineered an additional Fluc vector containing the same luciferase amino-acid sequence but with a nucleotide sequence of non-optimal codons to obtain a low CAI compared to the CAI spectra in human transcripts (Figure 4A and B, and Online Supplementary Figure S4B). As expected, the Fluc vector with a low CAI was less expressed than its high CAI counterpart under shSCR conditions (Figure 4C and Online Supplementary Figure S4C). After the induction of shRPS14 640 or shRPS14 641, and normalization to Rluc,
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haematologica | 2021; 106(3)