Figure 3. Validation of smORF translation by tagging assay.
(A–D) Ribosome footprints from small polysomes (pink) and mRNA reads (grey) mapped to smORFs, along with transcript and ORF models of (A) CG7630, (B) CG33774, (C) CR30055 (ncRNA), and (D) FBtr0072084_1 (uORF). Corresponding transfection assays in S2 cells are shown (FLAG antibody: green, F-actin stained with phalloidin: red, scale bars = 5 μm) together with Poly-Ribo-Seq metrics (RPKM, coverage and TE). Distribution of each peptide (reticular, other cytoplasmic or limited) is indicated.
Figure 3—figure supplement 1. Validation of smORF translation by tagging assay.
(A) Schematic of the transfection construct into which smORF 5′-UTRs and ORFs (no stop codon) were cloned under the Actin promoter, such as to be fused in frame to a C-terminal FLAG tag, with its own AUG start codon mutated to GCG. (B) Transfection negative controls, plasmid with no ORF (nor AUG), plasmid with the full-length tal transcript (minus 3′-UTR) with ORF-B tagged with FLAG, which has previously been shown not to be translated (Galindo et al., 2007), and a plasmid containing a putative smORF that is transcribed but not translated according to our Poly-Ribo-Seq (Uhg2-ORF1). (C) Immunoblot showing translation of FLAG-tagged smORFs (Table 3) corresponding to predicted sizes, along with β- tubulin loading control. (D) Different subcellular localisations of FLAG-tagged smORFs (green) corroborated by double staining with Mitotracker Red (red): “mitochondrial”, “other cytoplasmic” and “limited” (scale bar = 5 μm). (E) Correlation analysis of colocalisation between FLAG-tagged smORF peptides and Mitotracker Red, error bars represent SD from three experiments. (F) 50% of S2-cell translated smORFs show function in previous RNAi screens (Flymine). (G) Translation of FLAG-tagged pncr009:3L (ncRNA) ORFs 1, 2, and 3 in transfection assay with translational metric values shown (FLAG antibody: green, F-actin stained with phalloidin: red, scale bars = 5 μm). (H) Immunoblot showing detection of FLAG-tagged ORFs from pncr009:3L and CR30055 with predicted sizes (Table 4), along with β-tubulin loading control. (I) Translation of FLAG-tagged uORFs FBtr0072210_1 and FBtr0081720_1 in transfection assays with translational metric values shown (FLAG antibody: green, F-actin stained with phalloidin: red, scale bars = 5 μm).
Figure 3—figure supplement 2. Poly-Ribo-Seq reveals translation of ORFs in ncRNAs.
(A) Read density plot showing phasing of ribosome footprinting reads in the frame of smORFs within CR30055 and pncr009:3L detected as translated and confirmed by FLAG immunofluorescence translation assay. (B) Correlation of reads obtained by ORFs after Poly-Ribo-Seq (y axis) with reads obtained by sequencing of polysomal fractions before ribosome footprinting (x axis). The correlation is much stronger for canonical long ORFs and putative smORFs (grey) than for ncRNA ORFs (red). Many ncRNA ORFs below the 11.8 RPKM cut-off used to ascertain translation (green dotted line) can show association with polysomes (high Polysomal RNA RPKM), thus translation of ORFs in ncRNAs does not simply stem from non-coding association with polysomes.