Fig. 4: Evolutionary conservation of TERT and TUG1 intron retention.

a Alignment of human and mouse TUG1 locus (left) and TERT locus (right). Exons depicted with gray arrows, introns with magenta arrows. Below the alignment is shown a conservation map of conserved (red) and non-conserved (black) nucleotides. b UCSC Genome Browser showing poly(A)⁺ RNA-seq coverage from mouse embryonic stem cells (mES) across the Tug1 locus and Tert locus. Below, percentage intron retention (PIR) of Tert (right) and Tug1 (left) in mouse iPS (miPS) and mES cells obtained with vast-tools analysis on poly(A)⁺ RNA-seq. Values from human iPS (hiPS) cells are plotted for comparative purposes. Bars indicate means across replicates and dots individual replicates, n = 2 (miPS), 3 (mES). c Maximum intensity projections of representative images of Tug1 exon/intron smRNA FISH on mES cells. Exon in gray, introns 1 and 2 in magenta. Nucleus in blue, outlined with a dashed line. Scale bar, 5 μm. Middle: quantification of total and unspliced transcripts for each intron in the nucleus (N) and cytoplasm (C), solid line represents the mean. On the right: percentage of nuclear intron retention (PIR) for intron 1 and intron 2. Midline line, median; lower and upper box limits, 25th and 75th percentiles; whiskers, 1.5 times interquartile range from the 25th and 75th percentiles, n = 44 cells (intron 1), 30 cells (intron 2). d Relative subcellular localization of Tert and Tug1 in poly(A)⁺ RNA-seq from chromatin, cytoplasm and nucleoplasm of mES cells. Cytoplasm-enriched Gapdh and Actb and chromatin-enriched Firre are plotted for comparison. e Intron retention of TERT in seven mammalian species. Exon–intron structure is shown and scale bars indicate relative size for each species. Median intron retention across 38 (chimpanzee)—151 (mouse) cell and tissue types is represented by a color scale. Note high retention of introns 2 and 3 in opossum. Dashed lines indicate boundaries of orthologous introns that are retained in both species. Evolutionary relationships are represented by the cladogram on the right. Silhouettes from http://phylopic.org. f Cumulative distribution of maximum PIR levels for each coding and lncRNA gene in hiPS, mES and miPS cells (in purple). Minimum PIR value for the same gene is plotted in gray at the same x-axis position. Introns with maximum and minimum PIR values from TERT and TUG1 are connected with a yellow line.