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. 2022 Jan 5;11:e72867. doi: 10.7554/eLife.72867

Figure 5. Sm and CHTOP methylarginine mediates PRMT-dependent RI.

(a) Comparison of ΔΨ for RI following SNRPB knockdown (kd), GSK591 treatment, MS023 treatment, and CHTOPkd where ΔΨ=Ψ (Treatment)–Ψ (Control). (b, c) Comparison of ΔΨ z-score for common RI between SNRPBkd and CHTOPkd with either GSK591 (b) or MS023 (c) treatment. (d) Spearman rank correlation of ΔΨ for common RI between SNRPBkd and CHTOPkd with either GSK591 or MS023 treatment. (e) Schematic of Sm expression constructs where lollipops represent individual methylarginines. (f) RT-qPCR of RI following transduction with Sm R-to-A or R-to-K mutants relative to wild-type (WT). Data are represented as mean ± SD. Significance determined using Student’s t-test; *<0.05, ***<0.001, ****<0.0001. (g) Schematic of CHTOP expression construct as in panel (e). (h) RT-qPCR of RI following transduction with CHTOP R-to-A or R-to-K mutants relative to WT. Data are represented as mean ± SD. Significance determined using Student’s t-test; ***<0.001, ****<0.0001, ns=not significant.

Figure 5.

Figure 5—figure supplement 1. CHTOP and Sm arginine mutants phenocopy PRMT inhibition.

Figure 5—figure supplement 1.

(a) Comparison of ΔΨ for RI following ALYREFkd and CHTOPkd where ΔΨ=Ψ (Treatment)–Ψ (Control). (b) RT-qPCR of Exogenous Sm R-to-A and R-to-K mutants normalized to wild-type (WT) expression vector. Data are represented as mean ± SD. (c) Western blot of total cell extract from A549 cells expressing CHTOP or Sm WT, R-to-A, or R-to-K mutants. DB71=Direct Blue 71 membrane stain. See Figure 5—figure supplement 1—source data 1. (e) RT-qPCR of Exogenous CHTOP R-to-A and R-to-K mutants normalized to WT expression vector. Data are represented as mean ± SD. (f) RT-qPCR of Endogenous CHTOP in A549 cells expressing CHTOP WT, R-to-A, or R-to-K mutants. Data are represented as mean ± SD.
Figure 5—figure supplement 1—source data 1. Western blot data for Figure 5—figure supplement 1c.
Figure 5—figure supplement 2. Sm methylarginine is dispensable for snRNP assembly.

Figure 5—figure supplement 2.

(a) Western blot analysis of FLAG-targeted immunoprecipitation in A549 cells expressing Sm wild-type (WT), R-to-A, or R-to-K mutants. DB71=Direct Blue 71 membrane stain. See Figure 5—figure supplement 2—source data 1. (b) Northern blot analysis of snRNAs from FLAG- or SNRPB-targeted immunoprecipitation in A549 cells expressing Sm WT, R-to-A, or R-to-K mutants. See Figure 5—figure supplement 2—source data 2. (c) Northern blot analysis of snRNAs from SNRPB-targeted immunoprecipitation in A549 cells treated with DMSO, GSK591, or MS023 for 7 days. See Figure 5—figure supplement 2—source data 3. (d) Indirect immunofluorescence for FLAG (top/red) or SNRPB (bottom/green) in A549 cells expressing Sm WT, R-to-A, or R-to-K mutants. DAPI-nuclear stain is blue. See Figure 5—figure supplement 2—source data 4. (e) Indirect immunofluorescence for SNRPB (green) in A549 cells treated with DMSO, GSK591, or MS023 for 2 days. DAPI-nuclear stain is blue. See Figure 5—figure supplement 2—source data 5.
Figure 5—figure supplement 2—source data 1. Western blot data for Figure 5—figure supplement 2a.
Figure 5—figure supplement 2—source data 2. Northern blot data for Figure 5—figure supplement 2b.
Figure 5—figure supplement 2—source data 3. Northern blot data for Figure 5—figure supplement 2c.
Figure 5—figure supplement 2—source data 4. Image data for Figure 5—figure supplement 2d.
Figure 5—figure supplement 2—source data 5. Image data for Figure 5—figure supplement 2e.