Figure 3. uORFs reduce CDS translational divergence between D. melanogaster and D. simulans.

(A) The correlation of uORF TEs and the corresponding CDS TEs in 10 samples of Dm (D. melanogaster) and Ds (D. simulans). The bars represent Spearman’s correlation coefficient (rho). In all samples, we obtained both p-values <0.001. Data for the female head sample of Dm and Ds are shown as examples in the right panel. (B) The absolute values of interspecific TE changes for CDS and uORF in each sample between two species. For visualization purposes, all values greater than 4 were assigned a value of 4. ***, p<0.001, Wilcoxon rank-sum test. (C) Genes expressed in female heads (mRNA RPKM >0.1 in both species) were classified into three classes according to whether a gene had a conserved and dominantly translated uORF or not. Boxplots showing interspecific CDS TE variability |${\displaystyle lo{g}_2({\beta }_c)}$| of different gene classes. p values were calculated using Wilcoxon rank-sum tests between the neighboring groups. ***, p<0.001. (D) Genes expressed in female heads were classified into three classes according to the length of translated uORFs. Boxplots showing interspecific CDS TE variability |${\displaystyle lo{g}_2({\beta }_c)}$| of different gene classes. p values were calculated using Wilcoxon rank-sum tests between the neighboring groups. ***, p<0.001.

Figure 3—figure supplement 1. The positive correlation of interspecific TE changes between uORFs and CDSs.

Correlations between interspecific uORF TE changes (${\displaystyle \mathrm{l}\mathrm{o}{\mathrm{g}}_2{\beta }_u}$) and CDS TE changes (${\displaystyle \mathrm{l}\mathrm{o}{\mathrm{g}}_2{\beta }_{\mathrm{C}}}$) in 10 samples. The x-axis was divided into 50 equal bins with increasing ${\beta }_u$. Spearman’s correlation coefficients (rho) are shown at the top left. ***, p<0.001 in the correlation test.

Figure 3—figure supplement 2. Gene ontology analysis of the genes with ${\displaystyle lo{g}_2({\beta }_2)\ne 0}$ in each stage and tissue.

The biological process (BP) terms with q-values <0.05 in each sample type are indicated with red, and others are indicated with white.

Figure 3—figure supplement 3. Reads count distribution of uORFs and CDSs.

Distribution of mRNA read counts and RPF counts mapped to uORFs and CDSs for all expressed uORFs (A) or only highly expressed genes (B) in female head sample. The distribution patterns were similar across other samples, and the data are not shown here. Dm, D. melanogaster; Ds, D. simulans. The read counts were log₂ transformed.

Figure 3—figure supplement 4. The scheme illustrating the calculation of ${\beta }_u$, ${\beta }_c$ and $\gamma$.

When the cellular environment causes mRNA translation to be enhanced from baseline level, the TE_uORF changed from 2 to 4 (${\displaystyle {\beta }_u=2}$), while the TE_uORF changed with a smaller degree due to uORF’s buffering, from 4 to 6 (${\displaystyle {\beta }_C=1.5}$). This resulted in $\gamma$ <1. Conversely, when the cellular environment causes mRNA translation to be reduced from the baseline level, the TE_uORF changed from 2 to 1 (${\displaystyle {\beta }_u=0.5}$), while the TE_uORF changed with a smaller degree due to uORF’s buffering, from 4 to 3 (${\displaystyle {\beta }_C=0.76}$). This resulted in $\gamma$ >1. Overall, both ${\beta }_u$ >1 and $\gamma$ <1, and ${\beta }_u$ <1 and $\gamma$ >1, indicated the existence of uORFs’ buffering.

Figure 3—figure supplement 5. Conserved and dominantly translated uORFs showed the stronger buffering effect.

Genes expressed in different stages/tissues (mRNA RPKM >0.1 in both species) were classified into three classes according to whether a gene had a conserved and dominantly translated uORF or not. Boxplots showing interspecific CDS TE variability |${\displaystyle lo{g}_2({\beta }_c)}$| of different gene classes. p values were calculated using Wilcoxon rank-sum tests between the neighboring groups. ***, p<0.001.

Figure 3—figure supplement 6. Longer uORFs showed the stronger buffering effect.

Genes expressed in different stages/tissues (mRNA RPKM >0.1 in both species) were classified into three classes according to the length of translated uORFs. Boxplots showing interspecific CDS TE variability |${\displaystyle lo{g}_2({\beta }_c)}$| of different gene classes. p values were calculated using Wilcoxon rank-sum tests between the neighboring groups. ***, p<0.001.