Figure 1A. Scheme of the reporter library which includes random fragments of the transcriptome in the CDS. Transcripts share the same 5' and 3' UTR and 26 within the translation start site, which are recognized by a specific translation blocking morpholino (MO) (green). I, illumina adapters, purple. SP6, SP6 promoter. mRNAs are injected at the one cell stage in zebrafish and Xenopus, with or without a translation blocking morpholino. Reporter mRNA library is analyzed 8-9 hours post injection using high throughput sequencing. The analysis of variance (ANOVA) for the occurrence of each codon in the presence (-MO) or the absence (+MO) of translation is performed between triplicates in zebrafish and Xenopus to calculate the reporter Codon Stabilizing Index (rCSI). If codons cause differential mRNA stability in a translation dependent manner, potential stabilizing codons should be enriched in translated mRNAs (-MO vs +MO)(+rCSI), and destabilizing codons depleted (-MO vs +MO)(-rCSI).B. Boxplots showing the occurrence of the GGU and the CAC codons in the presence (+MO) and absence of MO in the reporter mRNA library 8 hours post injection (hpi) (triplicates).C. Biplots of the reporter codon stabilizing index (rCSI) in the presence and the absence of translation (+MO) in zebrafish and in Xenopusembryos, P < 2.2e-16 R=0.9, Spearman rank correlation. The rCSI is the ANOVA F-test value for the comparison of codon occurrences (e.g, panel B) between treatments in triplicates. This value is multiplied by -1 for depleted/destabilizing codons. D. Scheme of the codon stabilization coefficient (CSC) calculated as the Pearson correlation coefficient between the occurrence of each codon and the half life of each mRNA. Barplots ranking the 61 coding codons based on their codon stability coefficient (CSC). The encoded amino acid are indicated. E. Line plots of the relative demand of each codon in the zebrafish transcriptome at 6 hpf (% used), and the RNA levels for each tRNA (RPM). The P-value indicates the significant correlation with the CSC. F. Biplots of the rCSI comparing 8h vs 8h+MO and CSC in Zebrafishembryos, P < 1.2e-4 R=0.4, Spearman rank correlation. Based on the consistent identification by both methods we defined: Optimal codons, highlighted in red and non-optimal codons in blue. The intensity of the color represents the strength (light, weak; dark, strong) based on different CSC values.G. Boxplot of tRNA levels (RPM) for optimal and non-optimal codons (P = 3e-03, Wilcoxon rank-sum test). H. Cumulative distributions of the half-life of the maternal mRNAs in the absence of zygotic transcription in zebrafish. Shown are all mRNAs (gray), mRNAs with the highest fraction (top quartile) of optimal codons and the lowest fraction of non-optimal codons(red), mRNAs with the highest fraction of non-optimal codons and the lowest fraction of optimal codons (blue), in frame (left) or 1 nucleotide out of frame +1 (right). (P= 1e-8, in frame; P= 0.13, 1 nt out of frame, Wilcoxon rank-sum test). I. Diagram of two transcripts that only differ in a single nucleotide (G in yellow) which creates a frame shift, making one transcript enriched in optimal codons, and the other in non-optimal codons. Time-coursenorthern blot shows that non-optimal transcripts decay faster than their optimal counterparts. The radioactive intensity is shown in italics and normalized to the 1hpi sample for each injection.