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. 2019 Jul 8;8:e46327. doi: 10.7554/eLife.46327

Figure 2. RBPMS regulates AS in PAC1 cells.

(A) Schematic of experimental design of RBPMS knockdown and overexpression in PAC1 cells. (B) Western blots for RBPMS in PAC1 knockdown, left, and inducible lentiviral overexpression, right. FLAG antibodies were also used for the overexpression of 3xFLAG tagged RBPMS. GAPDH and TUBULIN were used as loading controls. (C) MA plots of alterations in mRNA abundance in PAC1 dedifferentiation, left, RBPMS knockdown, middle, and RBPMS A overexpression, right. Dark gray: genes with significant changes (p-adj <0.05). Light gray: genes with p-adj ≥0.05. Red: Rbpms, Rbpms2 and the SMC marker, Acta2. Numbers of up and down-regulated are shown at top and bottom. Horizontal lines; log2 fold change = 1 and −1. (D) AS changes (FDR < 0.05 and ΔPSI greater than 10%) in PAC1 cell dedifferentiation, left, RBPMS knockdown, middle, and RBPMS A overexpression, right. ASE were classified into skipped exon (SE), mutually exclusive exon (MXE), alternative 5′ and 3′ splice site (A5SS and A3SS) and retained intron (RI) by rMATS. Numbers indicate the number of significant ASE of each event type between the conditions compared. (E) Sashimi plots of selected ASEs. Ptprf is shown as a differentiated cassette exon (green), Piezo1 as a proliferative cassette exons (blue) and Actn1 as a MXE. The numbers on the arches indicate the number of reads mapping to the exon-exon junctions. PSI values for the ASE are indicated for each condition. Values correspond to the mean PSI calculated by rMATS. In the case of the Actn1 MXE, the percent inclusion of the SM exon is shown (PSI-SM). Schematic of the mRNA isoforms generated by the alternative splicing are found at the bottom as well as the chromosome coordinates. (F) RT-PCR validation of ASEs from panel E. Values shown are the mean of the PSI ± standard deviation (n = 3). Statistical significance was calculated using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). See Figure 2—figure supplement 3 for more ASEs validated in the RBPMS knockdown and RBPMS A overexpression by RT-PCR.

Figure 2.

Figure 2—figure supplement 1. Principal Component Analysis of PAC1 RNAseq samples and overlap of genes regulated at the splicing and abundance levels.

Figure 2—figure supplement 1.

(A) Principal Component Analysis (PCA) based upon mRNA abundance variance of RBPMS knockdown (D Ctr and D KD) and overexpression (P Ctr and P OE) replicates. (B) Schematics of the different AS types detected by rMATS. (C) Overlap of the number of genes affected at the levels of mRNA abundance and AS in the PAC1 dedifferentiation and RBPMS knockdown and overexpression comparisons.
Figure 2—figure supplement 2. AS changes of SM genes are specific to RBPMS expression.

Figure 2—figure supplement 2.

(A) Validation of lack of regulated splicing in lentiviral control populations (P LV) upon doxycycline treatment. (B) Validation of a second RBPMS siRNA (KD2, Stealth siRNAs, Thermo Fisher Scientific (RSS363829): CGCUUCGAUCCUGAAAUCCCGCAAA). Western blot probing for RBPMS in differentiated PAC1 cells treated with one of the siRNAs. GAPDH was used as a loading control in the western blot. RNAs were extracted and RT-PCR carried out to validate the ASE previously characterized for KD1. In (A) and (B), values shown are the mean ± sd of PSI (n = 3). Statistical significance was calculated using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001).
Figure 2—figure supplement 3. ΔPSI values determined by RT-PCR and RNA-Seq show good agreement.

Figure 2—figure supplement 3.

(A) Validation of differentiated and proliferative cassette and mutually exclusive splicing events by RT-PCR for both RBPMS knockdown and overexpression. Schematics indicate the splicing isoform corresponding to the PCR product. Values shown are the mean ± sd of PSI (n = 3). Statistical significance was calculated using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). (B) PSI correlation between the estimated ΔPSI from the rMATS analysis of the RBPMS knockdown and overexpression RNAseq experiments and the observed ΔPSI from the RT-PCR validations of RBPMS experiments (n = 28). Black line indicates the linear regression model. For statistical significance, a Pearson correlation test was carried out in RStudio and the results are shown in the right corner of the plot. r2 is the correlation coefficient and the p-value indicates the statistical significance of the correlation.