Fig 2: PNCTR Antagonizes Splicing Regulation Function of PTBP1(A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing.(B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated).(F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping.(G) Effects in (F) presented as differences in percent-spliced-in values (??; Wang et al., 2008b) between experimental treatments and the corresponding controls. Positive ?? values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test.(H) CLIP-seq and iCLIP analyses show that PTBP1 forms physical contacts with an extensive array of YUCUYY and YYUCUY motifs in front of CHEK2 exon 8. Functional significance of the PTBP1 interaction sequence highlighted in gray was validated in the minigene experiment in Figures S6L and S6M.(I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test.(J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test.(K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test.(L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test.See also Figures S5 and S6 and Table S2.

Fig 3: Study of the RNA/Protein Interactions between pCharme and PTBP1/MATR3(A) pCharme RNA-FISH (red, upper panel) and co-staining pCharme (red)/MATR3 (green, middle panel) or pCharme (red)/PTBP1 (white, bottom panel) in differentiated C2C12 myotubes. Blue lines indicate the edge of the nucleus. DAPI, 4',6-diamidino-2-phenylindole. Magnification of the yellow box is shown on the right. Scale bars, 15 µm.(B) Selected 3D-rendered iso-surface views showing MATR3/PTBP1 occupancy on pCharme transcript.(C) Scatter dot blot representing the quantification of pCharme/MATR3/PTBP1 overlapping signals. 3D-Pearson’s correlation coefficient indicates the means ± SEM calculated from images shown in (A) from four (pCharme/MATR3) and three (pCharme/PTBP1 and MATR3/PTBP1) biological replicates. ***p < 0.001, unpaired Student’s t test.(D) Neighboring MATR3 and PTBP1 binding sites (MATR3 consensus sequence [CT][CT]TTTCT.TTT, as reported in Uemura et al. [2017] and PTBP1-CLIP data and consensus sequence T[CT]T[CT][CT], as reported in Xue et al. [2009]) were mapped on pCharme intron-1 (black line). RRM, RNA recognition motif.(E) Hypothetical RNP complex comprising MATR3, PTBP1, and pCharme assembled on intron-1 (orange line), based on the structural data available in the Protein Data Bank. RRM, RNA recognition motif; PRI, PTBP1 RRM interaction motif.

Fig 4: Subcellular distribution of SINEUP RNAs after overexpression of SINEUP RBPs. (A1, B1) Representative RNA FISH with immunofluorescence images of the subcellular distribution of SINEUP RNAs and SINEUP RBPs in cells overexpressing PTBP1 (+PTBP1) (A1) or HNRNPK (+HNRNPK) (B1). Images for cells co-transfected with EGFP and SINEUP-GFP vectors (left images, A1 and B1) were compared with cells transfected with SINEUP-GFP vector alone (right images, A1 and B1). Bars indicate 5 µm. (A2, A3, B2, B3) Quantitative comparison of SINEUP-GFP RNA distribution between cells overexpressing PTBP1 (A2, A3) or HNRNPK (B2, B3) and cells without overexpression (Cont.). Results for cells co-transfected with EGFP and SINEUP-GFP vectors (A2 and B2) and those transfected with SINEUP-GFP vector alone (A3, B3) are shown. SINEUP RNA signals were detected using Icy Spot Detector. The ratio of spots in the nucleus and the cytoplasm were compared between overexpression and non-overexpression of SINEUP RBPs. *P < 0.05 by Student's t-test. Data are means ± SD of at least 10 independent cell images.

Fig 5: Increased PTB association with mt tRNAThr in apoptotic cells. (A) Interaction of PTB and mt tRNAThr during apoptosis. PTB was immunoprecipitated from extracts (Ext) prepared from HeLa cells treated with staurosporine as indicated. Accumulation of PTB and PARP-1 (lanes 1–4) and IP of PTB (lanes 6–9) was monitored by western blotting. Caspase-cleaved PTB (c-PTB) and PARP1 (c-PARP) are indicated. Co-IP of mt tRNAThr and U1 snRNA was measured by northern blotting. (B) Caspase inhibition in apoptotic cells has no effect on PTB and mt tRNAThr association. PTB was immunoprecipitated from extracts prepared from HeLa cells treated with z-DEVD-FMK and staurosporine as indicated. Other details are identical to panel A.