Modeling simulation of uORF-mediated translation buffering.

(A) Model schema of the modified ICIER model (on the top); the parameters are listed in the box below the schema. (B) Heatmap showing the CVs of CDS TE (NEC) under different ICDS (x-axis) and IuORF (y-axis) combinations with a uniform distribution of Rin input and the downstream dissociation model. The left panels elicited by the dotted lines from specific squares of right heatmap were two examples showing the distribution of NEC under ICDS = 0.8 & IuORF = 0 (top panel, without uORF) and ICDS = 0.8 & IuORF = 0.4 (bottom panel, with uORF). (C) Heatmap showing CVs of CDS TE (NEC) under different IuORF (x-axis) and IuORF (y-axis) combinations with a uniform distribution of Rin input and the downstream dissociation model. The left panels elicited by the dotted lines from specific squares of right heatmap were two examples showing the distribution of NEC under LuORF = 2 & IuORF = 0.2 (top panel) and LuORF = 30 & IuORF = 0.2 (bottom panel). (D) Heatmap showing median δ [log2(ΔNEC/ΔNEU=)] under different ICDS (x-axis) and IuORF (y-axis) combinations with a uniform distribution of Rin input and the downstream dissociation model. The left panel elicited by the dotted line from a specific square of right heatmap was an example showing the distribution of δ under ICDS = 0.8 & IuORF = 0.2. The vertical dashed line indicated the median value of δ.

Conservation and translation of uORFs between D. melanogaster and D. simulans.

(A) Spearman’s correlation coefficients (Rho, represented by the bars) of conserved TEs between Dm (D. melanogaster) and Ds (D. simulans). ***, P < 0.001. Data for the female head sample is shown as an example in the right panel. The x- and y-axes represent the uORF TEs in Dm and Ds. (B) The median of TE of conserved and species-specific uORFs in each sample. Each dot represents the median TE of a sample for a specific uORF class. Data from the female head sample is shown as an example in the right panel. P values were obtained from Wilcoxon rank sum tests. ***, P < 0.001. (C) uORFs were ranked by decreasing TEs within each gene. The uORF with the highest TE within each gene was defined as the dominant uORF (#1). EC2” represents the second highest uORF TE and the same goes for EC3” and “>3”. Each dot represents the median TE of a sample. (D) Fraction of conserved uORFs among dominant uORFs and other translated uORFs in each sample. The paired samples in Dm and Ds were linked together. The P value was obtained by the paired Wilcoxon signed rank test. ***, P < 0.001. (E) Absolute values of the interspecific TE fold changes (log2TE-FC) of dominant uORFs and the other translated uORFs in each sample. The paired samples in Dm and Ds were linked together. The median value of each sample is shown. The P value was obtained via the paired Wilcoxon signed rank test. ***, P < 0.001. Data from the female head sample were used as an example in the right panel.

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) Correlations between interspecific uORF TE changes (log2βu) and CDS TE changes (log2βC) in 10 samples. The x-axis was divided into 50 equal bins with increasing βu. Spearman’s correlation coefficients (Rho) are shown at the top left. ***, P < 0.001 in the correlation 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 |log2(βc)| 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 |log2(βc)| of different gene classes. P values were calculated using Wilcoxon rank sum tests between the neighboring groups. ***, P < 0.001.

Numbers of genes showing different magnitudes of TE changes between uORFs and CDS at the interspecific level.

uORFs could reduce CDS translational fluctuation during Drosophila development.

(A) The CV of TECDS across 10 Dm (D. melanogaster) samples and 10 Ds (D. simulans) samples. The selected gene with uORFs translated (TE > 0.1) in at least one Dm sample but its homologous gene without translated uORF in Ds samples. Each pair of dots linked by a gray line represents a pair of homologous genes in Dm and Ds. ***, P < 0.001, Wilcoxon signed-rank test. (B) The CV of TECDS across 10 Dm samples and 10 Ds samples. The selected gene with uORFs translated (TE > 0.1) in at least one Ds sample but its homologous gene without translated uORF in Dm samples. Each pair of dots linked by a gray line represents a pair of homologous genes in Dm and Ds. ***, P < 0.001, Wilcoxon signed-rank test. (C) Within each Drosophila species, the CV of TECDS of genes with translated uORFs compared to genes without the translated uORFs. The P values are obtained by the Wilcoxon rank sum test. ***, P < 0.001.

The strong buffering uORF of bcd and it knockout.

(A) Multiple sequence alignment of the bcd uORF and partial CDS in D. melanogaster and 20 other Drosophila species. The uORF and CDS are boxed in green and purple, respectively. The start codons of the uORF and CDS are boxed in red. (B) The coverage of mRNA-Seq (top), Ribo-Seq (middle), and TEs (bottom) of the bcd uORF and CDS in 0-2 h embryos of D. melanogaster (red) and D. simulans (blue). The uORF and CDS are denoted at the lower panel with dark green triangles and purple boxes, respectively. The 2 dashed lines mark the CDS region. The uORF TE, CDS TE and their interspecific changes were labeled at the bottom. (C) Genotypes of WT and two uORF knock-out strains (uKO1 and uKO2) generated by CRISPR-Cas9 technology. The uORF is boxed in dark green, and the red ATG represents the start codon of the uORF in the D. melanogaster genome.

Knocking out the bcd uORF increases CDS translation and perturbs the transcriptome during D. melanogaster embryogenesis.

(A) Dual-luciferase assay for bcd WT uORF and mutated uORF. The reporter structures of the WT and uORF mutants are illustrated on the left. The uORF mutant sequence was the same as that in the fly mutant created with CRISPR-Cas9 technology. The relative activity of Renilla luciferase was normalized to that of firefly luciferase. Error bars represent the S.E. of six biological replicates. Asterisks indicate statistical significance (***, P < 0.001). (B) Two ribosome fractions (monosome and polysome) of 0-2 h embryos were separated in a sucrose density gradient. Relative RNA abundance in the monosome and polysome fractions was quantified by real-time quantitative PCR. (C) P-to-M ratio of bcd mRNA (bcd mRNA abundance in polysome fraction/bcd mRNA abundance in monosome fraction) at 25°C (left) and 29°C (right). The P-to-M ratios of mutants were normalized to WT controls at 25°C and at 29°C, respectively. Error bars represent the S.E. of six biological replicates. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., P > 0.05). (D) The number of DEGs in each stage and their intersection with each other at 25°C (top) and 29°C (bottom). (E) Gene ontology analysis of DEGs at 29°C in each stage. The biological process (BP) terms with q-values < 0.05 in each stage are indicated in red and others are indicated in white.

Knockout of the bcd uORF reduces offspring number and starvation resistance.

(A) Comparison of the hatching rates (%) of mutant and WT offspring (n=20, Wilcoxon rank sum test; ***, P < 0.001). (B) The offspring number per maternal parent in different crosses over 10 days at 25°C. Asterisks indicate significant differences between various crosses and crosses of WT females with WT males (n=20, Wilcoxon rank sum test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., P > 0.05). The different crosses were denoted as the x-axis labels. (C) The offspring number per maternal parent in different crosses over 10 days at 29°C. (D) Survival curves of WT and mutant adult flies of females (left) and males (right) under starvation conditions. The black line represents the WT, the red line represents the uKO1/uKO1 mutant, and the blue line represents the uKO2/uKO2 mutant. Asterisks indicate significant differences compared to the WT. (n=200, log-rank test; ***, P < 0.001; n.s., P > 0.05).

uORFs function as translational buffers in primates.

(A) Boxplots showing the TEs of conserved and species-specific uORFs between Hs (H. sapiens) and Mm (M. mulatta). Data for the brain is shown as an example. Wilcoxon rank sum tests. ***, P < 0.001. (B) Spearman’s correlation coefficient (Rho) of uORFs’ TE between humans and macaques. The Rho values in the brain, liver, and testis were shown as bar plots. ***, P < 0.001. Data for the brain is shown as an example in the right panel. (C) Correlation between interspecific uORF TE changes (log2βu) and corresponding CDS TE changes (log2βC) in three tissues. The x-axis was divided into 50 equal bins with increasing βu. (D) Genes expressed in brains were classified into three classes according to the total length of translated uORFs. Boxplots showing interspecific CDS TE variability |log2(βc)| of different gene classes. P values were calculated using Wilcoxon rank sum tests between the neighboring groups. ***, P < 0.001. (E) Genes expressed in brains (mRNA RPKM > 0.1 in both species) were classified into three classes according to whether a gene had a conserved and dominantly translated uORF (TE > 0.1) in both species or not. Boxplots showing interspecific CDS TE variability |log2(βc)| of different gene classes. P values were calculated using Wilcoxon rank sum tests between the neighboring groups. ***, P < 0.001. (F) Boxplot showing the coefficients of variation (CVs) of CDS TE among the 69 lymphoblastoid cell lines (LCLs). Expressed genes (mean mRNA RPKM > 0.1) were divided into 20 bins with increased mRNA expression levels. In each bin, the genes were divided into two fractions according to whether the gene had a translated uORF or not. Wilcoxon rank sum tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.