Fig 1: Model depicting how MRV infection leads to alterations in the host cell AS. In normal conditions (A), U5 main components EFTUD2, PRPF8, and SNRNP200 are translated in the cytoplasm and imported in the nucleus. The U5 snRNP levels are decreased due to the normal turnover of the complex, but this decrease is balanced by the import of the protein components and assembly of new U5 snRNP, allowing the functional U5 snRNP level to stay at equilibrium. Upon infection (B), expression of MRV viral protein µ2 in the cytoplasm disturbs the capacity of the cell to produce EFTUD2, PRPF8 and SNRNP200 protein through a yet to define mechanism. This shutdown prevents the cell from replenishing the diminution of the U5 snRNP due to the normal turnover, and functional U5 snRNP level diminishes. This affects the capacity of the cell to regulate its AS, and leads to AS changes observed during MRV infection.
Fig 2: Molecular consequences of SNRNP200, SRRM1 and SRSF3 silencing in 22Rv1 cell line. a) Basal phospho-AKT, phospho-ERK1/2 and phospho-JNK levels in SNRNP200-, SRRM1- and SRSF3 silenced 22Rv1 cells (si-SNRNP200, si-SRRM1 and si-SRSF3, respectively; 24 h; n = 3). Protein levels were normalized by total AKT, ERK and JNK protein levels. Representative images are shown in right panel. Protein data were represented as percent of scramble cells. b) Expression levels of selected transcripts in response to SNRNP200 (upper panel), SRRM1 (central panel) and SRSF3 (bottom panel) silencing (24 h) in 22Rv1 cells. Ratio between the expression of splicing variants is shown in bars with dotted pattern. c) Expression levels of KHDRBS1, SFPQ and U2AF2 in response to SNRNP200- and SRRM1-silencing in 22Rv1 cells. d) Expression levels of C-MYC, PTEN and TP53 in response to SNRNP200-, SRRM1- and SRSF3-silencing in 22Rv1 cells. mRNA levels were determined by qPCR and adjusted by normalization factor calculated from ACTB and GAPDH expression levels. Data were represented as percent of scramble-treated control cells (mean ± SEM). Asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001) indicate statistically significant differences between groups.
Fig 3: Expression of spliceosome components and splicing factors in prostate cancer (PCa) samples. (a–b) Comparison of mRNA levels of spliceosome components (a) and splicing factors (b) between formalin-fixed paraffin embedded (FFPE) samples from PCa samples and non-tumor adjacent regions (N-TAR) (n = 84) determined by a microfluidic-based qPCR array. Data represent the mean ± SEM of mRNA expression levels adjusted by normalization factor (calculated from ACTB and GAPDH expression levels) and standardized by Z-score. c-d) ROC curves of a subset of spliceosome components and splicing factors generated by Random Forest computational algorithm (c) followed by cross validation analysis (d) to distinguish between tumor and N-TAR samples. e) Association between the expression levels of selected spliceosome components and splicing factors (SNRNP200, SRRM1 and SRSF3) and clinical parameters (Gleason score, T-Stage, perineural and lymphovascular invasion) in the same cohort of FFPE samples (n = 84). Correlations are represented by mean (connecting line) and error bands (pointed line) of expression levels. Data of associations represent the mean ± SEM of mRNA expression levels adjusted by normalization factor (calculated from ACTB and GAPDH expression levels). f) Association between SNRNP200, SRRM1 and SRSF3 expression levels and biochemical PCa recurrence in 67 samples from FFPE cohort (samples from patients who underwent adjuvant radiotherapy were not included), calculated by Log Rank analysis (LR). mRNA levels were determined by a microfluidic-based qPCR array and adjusted by normalization factor calculated from ACTB and GAPDH expression levels. Asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001) indicate statistically significant differences between groups.
Fig 4: U5 core components are required for MRV modulation of cellular AS and are reduced during infection by µ2. (A) Validation of the KD efficiency for EFTUD2, PRPF8 and SNRNP200 by western blot in mock- and T3DS-infected L929 cells. (B) Difference in splicing (?PSI) between infected and mock L929 cells in control siRNA or EFTUD2, PRPF8, and SNRNP200 siRNA-treated cells. Respective standard deviation for the mock and infected cells were added when calculating the standard deviation for the ?PSI. n = 3, biological replicates, unpaired two-tailed Student's t-test (ns, P > 0.05; *P = 0.05; **P = 0.01) comparing each condition against the control siRNA. (C) Protein levels of U5 snRNP components EFTUD2, PRPF8 and SNRNP200 in mock, T3DS and T3DK infected L929 cells. The membranes were H2O2-inactived and probed again against actin (EFTUD2, U2AF35) or vinculin (PRPF8, SNRNP200) as a loading control; a representative loading control is shown. On the right, the cumulative results for three western blots are summarized in a bar graph. The U2 snRNP protein U2AF35 was probed as a control. n = 3, biological replicates, unpaired two-tailed Student's t-test (ns, P > 0.05; *P = 0.05; **P = 0.01) comparing infected cells against control cells. (D) Impact of the ectopic expression of µ2 on U5 components protein levels at 24h and 48h in 293T cells. The U2 snRNP protein U2AF35 was probed as a control. The membranes were H2O2-inactived and probed again against actin (EFTUD2, U2AF35) or vinculin (PRPF8, SNRNP200) as a loading control; a representative loading control is shown. (E) Splicing profiles (PSI) of the HNRNPA2B1, TBP, and the negative control SERBP1 AS minigene reporters upon expression of the different µ2 constructs at 48 h. PCR amplicons were resolved using capillary electrophoresis and quantified using relative fluorescence. n = 3, biological replicates, unpaired two-tailed Student's t-test (ns, P > 0.05; *P = 0.05; **P = 0.01; ***P = 0.001; ****P = 0.0001) against the GFP alone condition.
Fig 5: Cell proliferation assay in response to enzalutamide treatment combined with SNRNP200, SRRM1 and SRSF3 silencing. Proliferation rate of 22Rv1 (a) and LNCaP (b) cell line was measured after 24 h of SNRNP200-, SRRM1- and SRSF3-silencing in the presence (DHT) or absence (no DHT) of 5a-dihydrotestosterone with or without enzalutamide (ENZA; n = 4). Results were expressed as percentage referred to scramble vehicle-treated control with DHT (mean ± SEM). Asterisks (* p < 0.05; ** p < 0.01), dash (#p < 0.05) and dollar sign ($p < 0.05) indicate statistically significant differences compared to DHT of scramble, DHT of each condition and DHT+ENZA of scramble, respectively.
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