Additional file 13 of The Tug1 lncRNA locus is essential for male fertility

Additional file 13: Fig. S8. The 5’ region of human TUG1 contains a conserved ORF. (A) GWIPS-viz tracks for human TUG1 genomic locus (hg38) is shown. Global aggregate of ribosome occupancy (ribosome profile), RNA-seq (mRNA coverage), and evolutionary protein-coding potential (PhyloCSF) across the TUG1 locus is shown. ORF1 and ORF2 are outlined with red and gray boxes, respectively. Tracks surrounding both ORFs are zoomed in for clarity (bottom). (B) Scheme showing human ORF1 construct design. hORF1 (labeled with a 3xFLAG epitope tag prior the stop codon) with the 5’UTR was inserted into pcDNA3.1(+) and transfected into HeLa cells. 48 hours post-transfection, TUG1-BOAT-3xFLAG localization was analyzed by immunofluorescence (IF) (shown in C). (C) Maximum intensity projection of HeLa cells expressing human 5’UTR-hORF1-3xFLAG. Localization of 3xFLAG tagged TUG1-BOAT was assessed by immunostaining against the 3xFLAG (green). Nucleus was monitored by DAPI (blue) and mitochondria was monitored by immunostaining against mitochondrial membrane translocase TOM20 (gray). Bar plot shows localization analysis of TUG1-BOAT. Scale bar is 5 μm.