Fig. 8

In vivo and in silico characterization of the protein-coding potential of InSETTs and InSETGs. a The log₂ cytosol/nucleus ratio of the novel and annotated transcripts of MAML2 and ESYT2 in the presence of etoposide. Error bars indicate the SE based on a total of 6 technical corresponding to 2 biological replicates. All data are shown relative to the log₂ cytosol/nucleus ratio of a nuclear-localized vlincRNA which is set to zero. b, c Schematic diagram of in silico predicted major ORFs in the InSETTs formed by the inclusion of the novel cassette InSETes in the MAML2 and ESYT2 loci chosen for these experiments (see Additional file 1: Fig. S9 and S10 for more details), and positions of the in-frame fusions of the GFP and mCherry proteins in those ORFs. Blue arrows indicate predicted ORF1 and ORF2, orange boxes indicate the novel exons. Green and magenta triangles indicate the position where GFP and mCherry proteins were inserted. d, e Fluorescence microscopy images and flow cytometry analysis of 293FT cells transfected with vectors harboring the GFP/mCherry fusions for the MAML2 or ESYT2 novel transcript. Scale bar, 100 mm. 293FT cells without transfection and those transfected with vectors expressing either only GFP or mCherry serve as the negative and positive controls respectively for gating. Only one representative biological replicate is shown — for the results from the other 2 biological replicates see Additional file 1: Fig. S11c. f Flow cytometry analysis of 293FT cells transfected with vectors harboring the GFP/mCherry fusions for the MAML2 or ESYT2 novel transcript with and without the CMV promoter. Only one representative biological replicate is shown — for the results from all 3 biological replicates see Additional file 1: Fig. S12. g Quantitation of the results of 3 biological replicates of experiments shown in the panel f, error bars represent SD. Asterisks show significant differences per two-sided paired Student’s t test (p value < 0.05). h Signals from the Ribo-seq assays for the two biological replicates of K562 cells treated with etoposide for 36 h and one replicate of pooled etoposide- and imatinib-treated cells (see “ Methods” for more details) are shown. The top portion represents zoom-in view of the ORF1 and ORF2 for the novel transcript in the ESYT2 locus (InSETT-8) that was used for the GFP/mCherry fusions. i Percentage of GENSCAN-specific exons found in introns of annotated genes containing Ribo-seq signal in both biological replicates of the etoposide-treated cells. Source data are provided in Additional file 2: Table S20