Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.

Supplementary figure 2: a, Maximum intensity projections of representative images of TUG1 exon/intron smRNA FISH on human foreskin fibroblasts (BJ). Exon in gray, intron 1 and intron 2 in magenta. Nucleus in blue and outlined with a dashed line. Scale bar, 5 μm. Middle: quantification (n = 50 cells) of spliced and unspliced transcripts for each intron in the nucleus (N) and cytoplasm (C), solid line represents the mean. On the right: correlation between nuclear intron count and quantity of nuclear TUG1; intron 1 in black, intron 2 in magenta. b, Total percentage of intron retention (total PIR) of each intron and percentage of nuclear enrichment of TUG1 (nuclear TUG1 over total cell TUG1) across indicated cell lines. Each dot represents one cell, n = 50 cells, at least 2 independent RNA FISH stainings. Midline line, median; lower and upper box limits, 25th and 75th percentiles; whiskers, 1.5 times interquartile range (IQR) from 25th and 75th percentiles.

Supplementary figure 3:
Maximum intensity projections of representative images of TERT exon and intron 2 smRNA FISH on HeLa cells. Exon in gray, intron 2 in magenta. Nucleus in blue and outlined with a dashed line. Scale bar, 5 μm. On top shown TERT low-expressing cell, on bottom TERT high-expressing cell. Experiment was performed three independent times.
Supplementary figure 4: a, Alignments of donor and acceptor splice sites and surrounding regions between human and mouse TUG1 and TERT. Arrows and dashed lines indicate the splice site. Mouse Tug1 E2/I2 junction is downstream of the E2/I2 junction in human TUG1. b, Comparison of donor and acceptor splice site strength measured using maximum entropy (MaxEnt) of human and mouse TUG1 and TERT retained and constitutively spliced introns.
Supplementary figure 5: eCLIP and RBPs motifs across TERT introns. a, Heatmap colored by total intron covered by eCLIP peaks for each of the 127 RBPs with TERT intron binding. b, Motif instance matches in TERT introns. In each window, the color represents the maximum scoring motif match in the window as calculated by FIMO (motifs only shown with p < 1e-4).

Supplementary figure 6: RNA binding protein (RBP) occupancy is increased over retained TERT introns. a,
Total fraction of each intron window covered by eCLIP peaks. The 15 blocks of data represent the 15 TERT introns. Each block is then divided into windows, which are 40bp upstream and downstream of the 5' and 3' splice sites; intron interiors are partitioned into five windows. b, Heatmap colored by fraction of window covered by eCLIP peaks for each of the 127 RBPs with TERT intron binding.  Fig. 8e and UCSC browser displaying additional Sanger sequencing results of the intron-spanning RT PCR. Arrowheads: black, spliced product; red, unspliced product. Star: black, less abundant spliced isoform; red, unspliced form upon TMO treatment. Kb, kilobases. PCR products after transfecting TUG1 TMOs were examined on agarose gel at least three independent times. b, Maximum intensity projections of TUG1 exon and intron 1 or intron 2 smRNA FISH on HeLa cells transfected with control TMO and with TUG1 TMO1 and TMO2. Nucleus in blue, outlined with a dashed circle; exon, gray; intron, magenta. Scale bar, 5 μm. Middle, quantification of spliced and intron-retained TUG1 for each intron in the nucleus (N) and cytoplasm (C), solid line represents the mean. Right, correlation between nuclear exon and intron 1 and 2 in cells treated with TUG1 TMO1 and TMO2 (red) or control TMO (black). Intron 1, n (control) = 49 cells, n (TUG1 TMOs) = 81 cells; intron 2, n (control) = 45 cells, n (TUG1 TMOs) = 46 cells from two independent measurements. c, U-2 OS and HeLa transfection efficiency assessed by TUG1 TMO1 labeled with FITC intake (green). Scale bar, 100 μm. Monitoring of transfection efficiency with TMO FITC was performed for each transfection experiment (n > 10).
Supplementary figure 10: a, Telomere restriction fragment (TRF) assay of LN-18 cells treated with transfection agent only, control TMO or TERT TMO up to 120 h. Sizes are shown in base pairs, dots indicate the mean telomere size for each lane. b, Cell viability and cell cycle analysis of HEK293T and LN-18 cells 72 h after transfection with TUG1, TERT or control TMO relative to control TMO. Bars, means across replicates; dots, individual replicates, error bars, standard deviation of the mean of three independent measurements. c, Quantification of γH2A.X foci of experimental conditions shown in b. Representative images of γH2A.X immunofluorescence are shown, DAPI, blue; γH2A.X, green; scale bar 5 μm. HEK293T, n (control) = 52 cells, n (TUG1 and TERT TMOs) = 50 cells; LN-18, n (control) 51 cells, n (TUG1 and TERT TMOs) = 50 cells. White circles, median; box limits indicate the 25th and 75th percentiles; whiskers, 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. For b and c P values were obtained by unpaired two-tailed ttest (equal variances), n.s. = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. In c, adjustments for multiple comparisons were not made.
Supplementary figure 11: Synthesis of thiomorpholino oligonucleotides. Synthesis of thiomorpholino oligonucleotides. Morpholino nucleosides 5-8 and morpholino phosphorodiamidites 9-12 were synthesized starting from appropriately protected ribonucleosides 1-4 as shown in the top part of the figure. The synthesis cycle begins with detritylation of succinyl CPG 500 supported nucleoside (A). Condensation with phosphorodiamidites 9-12 generates B, which is sulfurized to produce the thiophosphoramidate morpholino triester (C). After capping the failures, detritylation is carried out to produce (D), which is then ready for the next synthesis cycle. Cleavage of final TMO oligonucleotide was carried out using 33% aqueous ammonia at 55°C for 16 h.