Fig 2: NF-?B is involved in SRSF3-regulated miR-1908 expressionA. U2OS cells were transfected with control (CTRL) or SRSF3-specific siRNA. Transfected cells were resuspended into 12-well plates and then transfected with the NF-?B reporter vector. Transactivation of NF-?B was assessed by calculating the luciferase activity. B. To check whether NF-?B regulates the expression of miR-1908, cells were treated with the NF-?B-specific inhibitor BAY11-7082 (10 µM) for 24 h. The level of miR-1908-3p/5p was determined by RT-qPCR analysis using TaqMan primers. C. Cells were transfected with control (CTRL) or SRSF3-specific siRNA for 48 h and then whole cell lysates were prepared. Western blot analyses were performed using adequate specific antibodies. D. Cells were transfected as described above and then cellular fractionation was performed. Western blot analysis was performed to determine the localization of NF-?B (p65) and a-tubulin and lamin B was checked for cytoplasmic and nuclear markers, respectively. E-F. Cells were transfected with control (CTRL) or TAK1-specific siRNA and then the levels of TAK1 and phosphorylated IKKa/ß and NF-?B were assessed by Western blot analysis. The level of miR-1908-5p was determined by RT-qPCR analysis. G-H. The protein and mRNA expression levels of NF-?B target genes including HIF1-a, cyclin D1, and slug, were determined by western blot and RT-qPCR analysis, respectively. All experiments are performed more than three times and data represent the mean ± S.D. Asterisk (*) indicates statistical significance of p<0.05 as determined by Student's t-test.

Fig 3: SRSF3 represses p21 translation though interacts p21cip1/waf1 3'UTR region.A RNP-immunoprecipitation (RNP-IP) analysis was performed using a IgG (mouse) or an anti-SRSF3 antibody on cytoplasm extract from SW480 cells, followed by RT-PCR and western blot analysis. B HEK293T cells transfected with Flag-SRSF3. RNP-IP analysis was carried out by using the anti-Flag antibody, followed by RT-PCR and western blot analysis. C SW480 cells treated with siControl or siSRSF3 siRNA. RNP-IP analysis were carried out using an IgG or an anti-eIF4A1 antibody. It was followed by western blot and RT-PCR analysis. D A schematic of firefly luciferase constructs in which the 5'UTR and 3'UTR of p21cip1/waf1 were cloned upstream or downstream of the luciferase coding region (Luc.). E HEK293T cells were transfected with pGL3-vector, pGL3-p21cip1/waf1 5'UTR, and pGL3-p21cip1/waf1 3'UTR plasmids and subjected to RNP-immunoprecipitation analysis as described for panel. F Luciferase assays were performed after transfecting pGL3-vector, pGL3-p21 5'UTR and pGL3-p21 3'UTR construct into SW480 cells transfected with either siControl or siSRSF3. Renilla luciferase vector was co-transfected for normalization.

Fig 4: SRSF3 interacts with eIF4A1 in the cytoplasm.A The siControl or siSRSF3 treated SW480 cells were detected SRSF3, p21cip1/waf1 and translation initiation factor proteins. The protein, ß-actin was used as a loading control. B HEK-293T cells transfected with Flag-eIF2a, Flag-eIF5A, Flag-eIF3F, and Flag-eIF4G1. Co-immunoprecipitation was carried out using the anti-Flag antibody or eIF4A1 antibody. Levels of indicated proteins in cytoplasmic extract and Co-IP products were analyzed by western blot analysis. C Cytoplasmic extract of SW480 cells were immunoprecipitation with an anti-SRSF3 or anti-eIF4A1 antibody. The immunocomplexes and input were analyzed using western blot with anti-Flag, anti-SRSF3, anti-rpS3, and anti-rpL13a antibodies. D U2OS cells were treatment with or without sodium arsenite (0.5 mM, 1 h) for the mentioned time period before for immunofluorescence microscopy. Cells were stained with eIF4A1 (Green), SRSF3 (Red). Scale bar, 10 µm. E Schematic representation of full length and a series of deletion mutants of SRSF3. F Lysates from HEK293T cells transfected with Flag-tagged full length (FL) or deletion mutant SRSF3 were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis with anti-Flag, anti-eIF4A, and anti-ß-actin antibodies. G In vitro GST-pulldown assay of the binding of recombinant Hisx6-tagged eIF4A1 with GST or the GST-SRSF3 (FL, RRM, and RS domain).

Fig 5: Cellular mTOR activity affects the expression profile of U2af1 isoform. (A) U2af1a is selectively up-regulated upon mTOR activation. (left) RNA-Seq reads alignments of U2af1 isoforms in WT and Tsc1-/- transcriptomes. (right) Quantitation of U2af1 isoforms in the RNA-Seq data from WT and Tsc1-/- MEFs. (B) Expression of U2af1 isoforms in WT and Tsc1-/- MEFs was measured by Taqman qPCR with absolute quantitation. The data are presented as the mean (SD) (*P = 1.3e-6, **P = 0.60; two-tailed Student's t test, n = 3 for technical repeats). Western blot analyses of U2AF1 isoforms were done using total cellular extracts from WT and Tsc1-/- MEFs. Please note that U2AF1b isoform is only visible in the longer exposure blot. Phospho-S6 (pRPS6) probing is for the validation of mTOR activation. SE and LE indicate short exposure and long exposure in western blot, respectively. (C) U2af1a is selectively up-regulated upon the activation of cellular mTOR signaling. (left) A workflow of the serum add-back experiment for the manipulation of cellular mTOR activity. (right) Absolute quantitation of U2af1 isoforms by Taqman qPCR. The data are presented as the mean (SD) (*P = 1.6e–4, **P = 0.43; two-tailed Student's t test, n = 3 for technical repeats). (D) A workflow of the screening strategy for mTOR-regulated splicing factors that regulate the U2af1 isoform expression. DGE, differential gene expression at the transcript level. (E) An RNAi screen to identify a regulator(s) of U2af1 alternative splicing. SR splicing factors were knocked down by siRNAs in Tsc1-/- MEFs and the expression of U2af1 isoforms was measured by Taqman qPCR assay with absolute quantitation. Asterisks denote statistically significant changes of U2af1 isoform expression upon the RNAi knockdown. The data are presented as the mean (SD) (*P < 0.0086; two-tailed Student's t test, n = 3 for technical repeats). (F) Western blot analysis of SRSF3 in WT and Tsc1-/- MEFs. TUBULIN and HNRNPA1 were used as loading controls. Quantitation by ImageQuant software of the SRSF3 signals normalized to TUBULIN or HNRNPA1 is shown on the right. (*P < 7.5e-4; two-tailed Student's t test, n = 3 for biological repeats; see Supplementary Figure S1N for the other two repeats). (G) Relative expression of U2af1a, U2af1b and U2af1c transcripts (structure shown on right; PTC, premature termination codon) upon RNAi knockdown of Srsf3. Puromycin was added for 8 h at the concentration of 5 µg/ml. The data are presented as the mean (SD) (*P <0.010, **P = 0.31; two-tailed Student's t test, n = 3 for technical repeats). (H) A proposed model for regulation of U2af1 tandem exon splicing by mTOR and SRSF3.