The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

Abstract

Protein arginine methyltransferase 5 (PRMT5) is a promising cancer target, yet it is unclear which PRMT5 roles underlie this vulnerability. Here, we establish that PRMT5 inhibition induces a special class of unspliced introns, called detained introns (DIs). We used the depletion of CLNS1A, a PRMT5 cofactor that specifically enables Sm protein methylation, to interrogate the impact of DIs. We found that the disruption of Sm protein methylation is sufficient to induce DI upregulation, cell cycle defects, and loss of viability. Finally, we discovered that PRMT5-regulated DIs, and the impacted genes, are highly conserved across human, and also mouse, cell lines but display little interspecies conservation. Despite this, human and mouse DIs have convergent impacts on proliferation by affecting essential components of proliferation-regulating complexes. Together, these data argue that the PRMT5-splicing axis, and particularly appropriate DI splicing, underlie cancer's vulnerability to PRMT5 inhibitors.

Keywords: Cancer; Gene network; Genomic analysis; Transcriptomics.

Graphical abstract

Figure 1

PRMT5 inhibition causes the formation of DIs specifically in the nucleus (A–C) U87 cells were treated with vehicle or 10 nM JNJ-64619178 (PRMT5i) for 3 days and polyA + mRNA was deep sequenced and analyzed to identify and quantify exon-based alternative splicing (AS) events, including DIs, as shown in the schematic (A). (B) Dots represent individual events for each AS type, with color denoting those significantly (p_adj < 0.05) or not significantly (gray) altered by treatment. (C) Proportion of each AS event type that is significantly (p_adj < 0.05) altered by treatment. The total number of significant AS events is indicated above each bar. (D–F) U87 cells were treated with vehicle or PRMT5i (as above). Nuclear and cytoplasmic fractions were isolated prior to sequencing (as above). (D) Heatmap shows intron-containing isoforms that are significantly up- or down-regulated by PRMT5i-treatment in either nuclear or cytoplasmic compartments (p_adj < 0.05). Columns show replicates, rows indicate individual introns, and the coloring indicates Z score normalized isoform counts where red denotes higher expression and blue denotes lower expression. (E) For all PRMT5-regulated DI isoforms (Left), and their coding isoforms (Right). The y axis values show the proportion of reads present in nuclear versus cytoplasmic fractions (1 – exclusively nuclear, 0 – equal distribution, −1 – exclusively cytoplasmic). The x axis values show the proportion of reads in vehicle versus PRMT5i-treated samples (1 – exclusively in PRMT5i samples, 0 – equal distribution, −1 – exclusively in vehicle samples). Each dot represents a specific isoform. (F) Representative sequencing tracks for a DI (red), and its adjacent exons (gray), in DNA2 in nuclear or cytoplasmic fractions and in whole cell lysates, with or without PRMT5i treatment. (G) Comparison of the induction of PRMT5-regulated DIs (Left) and their coding isoforms (Right) detected in nuclear (DIs) or cytoplasmic (coding) fractions versus whole cell lysate. Inset shows the region displayed in the larger graphs. Correlation values reflect the Pearson correlation coefficient. (H) Bar graphs (Top) show the quantification of DI (red) and coding (gray) isoform levels for six representative PRMT5-resposive genes (as indicated), determined by qRT-PCR of total RNA from whole cell lysates of U87 cells treated with vehicle or 10 nM JNJ-64619178 (PRMT5i) for 3 days. Data are mean ± SD of 3 technical replicates. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, Student’s t test. Protein levels (Bottom) were determined by western, using GAPDH as a loading control. Quantifications are shown below each blot. For this and all subsequent western blots, numbers indicate approximate molecular weight in kilodaltons.

Figure 2

PRMT5 regulates a conserved set of DIs across multiple human cell lines (A) Human cell lines used for AS analysis, including cancer or tissue of origin, driver mutations or construct, and MTAP status. (B) Proportion of each AS event type that show significantly altered levels in the indicated cell line in response to 3-day treatment with 10 nM JNJ-64619178 (PRMT5i) compared to vehicle control (p_adj < 0.05). The total number of significant AS events for that cell line is indicated to the right of each bar. (C) Relative expression of individual DIs across all human cell lines after 3-day vehicle or PRMT5i treatment. Each column represents a specific DI that is significantly up- or down-regulated by PRMT5i-treatment in at least one cell line. Coloring represents Z score normalized intron counts, where red indicates higher relative expression and blue indicates lower relative expression. (D) Similarity matrix of the indicated AS event significantly altered by PRMT5i treatment (p_adj < 0.05). Each square represents the similarity between the two intersecting cell lines, with coloring indicating percent similarity (dice similarity score) and square size indicating the number of similar events. (E) Bar charts of DI conservation across 7 human cell lines. Bar length represents the number of significant PRMT5i-upregulated DIs (p_adj < 0.05, log₂FC > 0) for each cell line and the color denotes the number of cell lines in which a given DI is conserved across. The total number of significant DIs in each line is indicated to the right of each bar. (F) Enriched GO terms from the KW Biological Process gene set for PRMT5i-upregulated DIs in each cell line (p_adj < 0.05, log₂FC > 0). Terms are displayed if they are significant in at least one cell line (FDR < 0.01). Color represents enrichment score, while size inversely correlates with significance value.

Figure 3

Generation of endogenous dTag-CLNS1A in multiple human cell lines to specifically inhibit the PRMT5-splicing axis (A) Schematic indicating how use of the dTag-CLNS1A degron system specifically separates the disruption of the methylosome from other PRMT5 functions. (B) Western blot analysis of representative dTag-CLNS1A clones for CAL51, HEK293T, and HCT116 cells, alongside parental controls with antibodies against CLNS1A, the inserted HA tag, or loading control GAPDH. Asterisk (∗) marks a non-specific band. (C) Dose-response curves for three parental cell lines treated with dTag-13 for 6 days. Data are mean ± SD of 3 technical replicates/line. Indicated p-values represent the most significant comparison between the respective drug concentration and vehicle-treated cells. Vertical dashed line represents the concentration above which dTag-13 has off-target effects. ∗∗∗∗p < 0.0001, Student’s t test. (D) Western blot analyses of HA (CLNS1A) and loading control GAPDH in four CAL51, HEK293T, and HCT116 dTag-CLNS1A clones treated with either vehicle or 1 μM dTag-13 for 3 days.

Figure 4

Loss of Sm protein methylation correlates with increased DI formation and decreased viability (A–C) dTag-CLNS1A clones for CAL51, HEK293T, and HCT116 were treated with vehicle, 10 nM JNJ-64619178 (PRMT5i), or 1 μM dTag-13 for 3 days (A) Western blot analyses of HA (CLNS1A), SDMA (methyl-SmB), and loading controls (GAPDH for CAL51 and HEK293T, or HSP90 for HCT116). (B) Quantification of HA (Left) and methyl-SmB (Right) levels normalized to loading control. Data are mean ± SD of 4 biological replicates. Welch’s t-test. (C) Quantification of indicated classes of AS event in dTag-CLNS1A #1 for CAL51, HEK293T, and HCT116. Dots represent individual events, denoting ones that are significantly (p_adj < 0.05, color), or not significantly (gray) altered by dTag-13 treatment. (D) Venn diagrams show overlap of DIs in CAL51, HEK293T, and HCT116 cells that were significantly altered by dTag-13 versus PRMT5i treatment, compared to their vehicle controls (p_adj < 0.05). Hypergeometric test. (E) CAL51, HEK293T, and HCT116 dTag-CLNS1A #1 clones were treated with vehicle, 10 nM PRMT5i, or 1 μΜ dTag-13. These were analyzed by FACS for: (Left graphs) cell cycle profile by DAPI/EdU staining after 4 days, or (Right graphs) cell death by Annexin-V/PI staining after 6 days of treatment. Cell cycle data represent mean ± SD of 4 biological replicates, and cell death data represent mean ± SD of 3 biological replicates. Corresponding statistics, and additional timepoints, are shown in Figures S5A, S5B, S6A, and S6B. (F) Relative viability of four dTag-CLNS1A clones versus parental lines for CAL51, HEK293T, and HCT116 cells after a 6-day dTag-13 treatment. Data represent mean ± SD of 3 technical replicates. Student’s t test. For all panels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 5

HCT116 cells are CLNS1A-independent until PRMT5 activity is decreased (A) Western blot analyses of HA (CLNSIA), SDMA (methyl-SmB), and HSP90 as loading control in HCT116 dTag-CLNS1A #1 (Left) and #2 (Right) maintained in 1 μM dTag-13 for the indicated number of days. (B) Representative Western blot analysis of CLNS1A, SDMA (methyl-SmB), and loading control GAPDH levels in HCT116 sgCtrl and four sgCLNS1A clones (n = 4 biological replicates). (C) Relative proliferation of HCT116 sgCtrl and four sgCLNS1A clones through analyses of cumulative population doubling over a 6-day period. Data represent mean ± SD of 10 technical replicates. (D) Relative viability of HCT116 sgCtrl and indicated sgCLNS1A clones treated with the PRMT5i JNJ-64619178 for 6 days. Data represent mean ± SD of 3 technical replicates. (E–G) HCT116 parental or two dTag-CLNS1A clones treated with or without 1μM dTag-13 in combination with the indicated concentrations of JNJ-64619178 (PRMT5i). (E) Relative viability was assessed after treatment for 6 days. Data represent mean ± SD of 3 technical replicates. Red markings correspond to the critical PRMT5i doses used in Figures 5F and 5G. (F) Western blot analyses of HA (CLNS1A), SDMA, and loading control HSP90 after treatment with the critical indicated PRMT5i doses for 3 days (G) Relative levels of a representative DI in EIF4E were assessed by qRT-PCR after treatment with the critical indicated PRMT5i doses for 3 days. Data represent mean ± SD of 3 technical replicates. For all panels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, Student’s t test.

Figure 6

PRMT5 regulates a conserved set of DIs in multiple mouse cell lines (A) Summary of mouse cell lines used for AS analysis, including cancer type, driver mutations, PRMT5i used, and MTAP status. (B) Proportion of each AS event type that is significantly different in indicated cell line after 3-day PRMT5i treatment compared to vehicle control (p_adj < 0.05). Total number of significant AS events is indicated above the bar. (C) Relative expression of individual DIs across all mouse cell lines after 3-day vehicle or PRMT5i treatment. Each column represents a specific DI that is significantly upregulated or downregulated by PRMT5i-treatment in at least one cell line. Colors represent column Z score normalized intron counts, where red indicates higher relative expression and blue indicates lower relative expression. (D) Similarity matrix of the indicated class of AS event that was significantly altered by PRMT5i treatment. Each square represents the similarity between the two intersecting murine cell lines. Square coloring indicates percent similarity (dice similarity score), and square size is proportional to the number of similar events. (E) Bar charts of DI conservation across all mouse cell lines. Each bar represents the number of PRMT5i-upregulated DIs (p_adj < 0.05, log₂FC > 0) in each cell line and the color corresponds to the number of cell lines in which a given DI is conserved across. Total number of significant DIs in each line is indicated to the right of the bar. (F) Enriched GO terms from the KW Biological Process gene set for PRMT5i-upregualted DIs in each cell line (p_adj < 0.05, log₂FC > 0). Terms are displayed if they are significant in at least one cell line (FDR < 0.01). Color represents enrichment score, while size inversely correlates with significance value.

Figure 7

PRMT5-regulated DI identity is not conserved across species, but it occurs in genes in similar complexes to regulate proliferation-associated processes (A) Overlap of common DI-regulated (Left) processes, (Center) genes, and (Right) complexes for PRMT5-regulated DIs in human (H23) and mouse (KP393T5) lung cancer cell lines. Percentages represent the dice similarity score for each comparison. (B) Enrichment of DI-regulated biological processes in human and mouse cells. Each pair of bars represents a specific biological process, with the blue bar representing the FDR of the human H23 lung cancer line and the red bar representing the FDR of the mouse KP393T5 lung cancer line. Vertical dashed line represents the significance cutoff (FDR < 0.05). (C) Schematic of representative DI-regulated complexes, including the minichromosome maintenance (MCM) and mediator (MED) complexes. Each shape represents a specific complex member, and a color fill of blue, red, purple, or white indicates that the complex member contained a DI in human, mouse, both species, or neither, respectively. (D) Sankey plot showing convergence of DI-regulated complexes and biological processes. Each line represents a specific DI moving from (Column 1) gene to (Column 2) complexes to (Column 3) biological processes. Line color signifies conservation at the (Left) gene or (Right) complex level, and thickness corresponds to the number of (Left) complexes or (Right) biological processes a given gene is a part of. Column color represents conservation at the level of (Column 1) gene, (Column 2) complexes, and (Column 3) biological processes. Blue indicates unique to human cells, red unique to mouse cells, and purple common to both species. For biological processes, white indicates DIs/complexes that map to non-significant biological processes (FDR > 0.05).