Fig. 5

The 5′ region of Tug1 encodes a conserved protein. a Open reading frame (ORF) search in human and mouse Tug1 reveals multiple ORFs (arrows). ORF1 and ORF2 (red arrows) indicate two ORFs with greater than 70% amino acid conservation between humans and mice (92% and 70%, respectively). b Tug1 mouse genomic locus (mm10) is shown. Ribosome occupancy (ribosome profile), RNA-seq (mRNA coverage), and evolutionary protein-coding potential (PhyloCSF) across the Tug1 locus in mouse embryonic fibroblasts (MEFs) are depicted. ORF1 and ORF2 are outlined with red and gray boxes, respectively (top). Tracks surrounding both ORFs are zoomed in for clarity (bottom). c ORF RNA in wild-type and Tug1^−/− testes for ORF1. Top: scheme of Tug1 locus showing the location of primers (triangles) and ORF1 (red rectangle). Bottom: RT-PCR of Tug1, Tug1-ORF1, and the endogenous control 7SK. d Scheme of human and mouse ORF1 construct design. A 3xFLAG epitope tag was inserted prior to the stop codon of ORF1. Mouse constructs were dual-tagged with both 3xFLAG and HA tags. Expression constructs were designed with (hORF1+UTR, mORF1+UTR) and without (hORF1, mORF1) the 5′ UTR. Constructs and GFP as control were inserted into pcDNA3.1(+). Forty-eight hours post-transfection, 3T3 and HeLa cells were harvested for western blot (WB) (shown in e) or analyzed by immunofluorescence (IF) (shown in f, g). e Western blot targeting the 3xFlag (FLAG) in 3T3 and HeLa cells expressing human and mouse constructs, respectively. GAPDH was used as a loading control. f, g Maximum intensity projections of 3T3 cells expressing human and mouse constructs. Immunostaining against the Flag tag (green) and DAPI (blue) is shown. The bar plot shows the localization analysis of human and mouse TUG1-BOAT. N & C indicates nuclear and cytoplasmic localization, and C indicates only cytoplasmic. Scale bar is 5 μm