uORFs within DAP5 translationally activated mRNAs are translated independently of DAP5. (A) A correlation between the uORF read count of the shNT samples and the shDAP5 samples as calculated by PRICE. Each data point represents an uORF, where the uORF read count was normalized to the read count of the main CDS. DAP5 translationally activated transcripts are colored in red filled circles. The dashed red and black lines are the linear regression fit to DAP5 activated uORFs and the remaining uORFs, respectively. Statistical significance between the two groups was determined by the ANCOVA test. (B) Heat map of the mean uORF/CDS ratio in control and DAP5 KD hESCs for DAP5 translationally activated targets identified by PRICE. Mean ratios were obtained from the uORF and CDS reads of the four Ribo-seq replicates, and were normalized to the mean shNT ratio, expressed as 1. Color scale indicates increasing mean uORF/CDS ratio, indicative of increasing DAP5-dependent translation of CDS as compared to uORF. uORFs with similar or lower mean ratios in DAP5 KD hESCs compared to NT hESCs correspond to uORFs that are likewise translated in a DAP5 dependent manner. Targets with more than one uORF identified are presented accordingly with individual uORF/CDS ratios. (C) Ribosome profiling footprints in NT (blue) and DAP5 (red) KD hESCs, and total mRNA reads (black, one representative NT KD sample) distribution along the 5′ leader and the 5′ proximal coding sequence of the indicated genes. Shown are snapshots from the UCSC Genome Browser with the predicted uORFs demarked by black lines of corresponding lengths with initiation codon indicated. Ribo-seq replicates are presented in track collection mode using merging method “add.” The structure of the mRNA is shown at the bottom. Chromosomal position is shown at top. For clarity, all genes are shown in the 5′ to 3′ orientation regardless of the chromosomal orientations of their respective loci; genes that are encoded on the minus strand are shown on the reverse strand (e.g., LGR4, PROSER1, ROCK1). Genes for which the 5′UTRs span more than a single exon are presented using “multi-exon” view. Arrows indicate initiation of CDS. Additional genes with manually detected uORFs that were missed by the PRICE platform are shown in Supplemental Figure S2.
