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. 2015 Sep 29:5:14548.
doi: 10.1038/srep14548.

PTBP1 and PTBP2 impaired autoregulation of SRSF3 in cancer cells

Jihua Guo  1 , Jun Jia  1 , Rong Jia  1
Affiliations

PTBP1 and PTBP2 impaired autoregulation of SRSF3 in cancer cells

Jihua Guo et al. Sci Rep. .

Abstract

Splicing factors are key players in the regulation of alternative splicing of pre-mRNAs. Overexpression of splicing factors, including SRSF3, has been strongly linked with oncogenesis. However, the mechanisms behind their overexpression remain largely unclear. Autoregulation is a common mechanism to maintain relative stable expression levels of splicing factors in cells. SRSF3 regulates its own expression by enhancing the inclusion of an alternative exon 4 with an in-frame stop codon. We found that the inclusion of SRSF3 exon 4 is impaired in oral squamous cell carcinoma (OSCC) cells. PTBP1 and PTBP2 bind to an exonic splicing suppressor in exon 4 and inhibit its inclusion, which results in overexpression of full length functional SRSF3. Overexpression of SRSF3, in turn, promotes PTBP2 expression. Our results suggest a novel mechanism for the overexpression of oncogenic splicing factor via impairing autoregulation in cancer cells.

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Figures

Figure 1
Figure 1. Autoregulation of SRSF3 expression is disturbed in OSCC cancer cells.
(A) SRSF3 is overexpressed in oral squamous cell carcinoma cell lines. Western blot analysis showed the expression of SRSF3 in primary human oral squamous carcinoma cells (T1 to T5), CAL 27 or normal primary oral mucosal epithelial cells (N1 to N3). β-actin served as loading control. (B) SRSF3 is required for the growth of oral squamous cell carcinoma cells. A total of 5 × 105 CAL 27 or T3 cells were inoculated into 6 well plates and transfected by 20 nM SRSF3 siRNA (#1) or non-specific siRNA with Lipofectamine 2000 on Day 0. Cells were passed and received another transfection on Day 2. Cell numbers were counted on Day 4. Western blot analysis showed knockdown efficiency of SRSF3. *p < 0.05. (C) Schematic diagram of the pre-mRNA and splicing pattern of human SRSF3. Human SRSF3 pre-mRNA contains an alternatively spliced exon 4 between exon 3 and exon 5. There is an in-frame stop codon in the 5′ end of exon 4. The numbers above the pre-mRNA exons (boxes) and introns (lines) are the nucleotide positions in pre-mRNA. Dashed lines above or below introns indicate RNA splicing direction. (D) Alternative splicing of exon 4 in OSCC cells (CAL 27, and T1-T3) or normal primary oral mucosal epithelial cells (N1 to N3) was analyzed by RT-PCR with primers located in the constant exon 3 and 7. Because SRSF3 transcript without exon 4 (S) is much more than that with exon 4 (L) in cells, an exon 3/5 forward junction primer was also used to specifically amplify exon 4-excluded short product. Relative L/S represents the ratio of band intensities of long vs short isoforms. GAPDH served as loading control. Diagrams on the right of (D) show the structures of human SRSF3 pre-mRNA and spliced products and primer positions (short lines above or below exons).
Figure 2
Figure 2. PTBP1 interacts with the ESS motif of SRSF3 exon 4.
(A) Diagram of SRSF3 minigene. Genomic sequence from 3′ part of exon 3 to 5′ part of exon 5 of SRSF3 was amplified from CAL 27 ells and cloned into pEGFP-N1. A kozak consensus sequence and an in-frame ATG were added to the 5′ end of minigene. GFP is allowed to be translated in the absence of exon 4. To mapping potential regulatory motifs, the 3′ part of alternative exon 4 was deleted or serially mutated. (B) Wild type (wt) or mutated (mt) minigenes were transfected into 293 cells. Alternative splicing of exon 4 were analyzed by RT-PCR. L/S represents the ratio of band intensities of long vs short isoforms. Neomycin resistant gene served as loading and transfection efficiency control. GFP fluorescent intensities in transfected cells were also analyzed by FACS. The diagram below neomycin showed the geometric means of green fluorescent intensities of minigene transfected cells (duplicate samples for each plasmid). (C) RNA pulldown assay showed that PTPB1 interacts with the ESS motif of SRSF3 alternative exon 4. Biotinylated oligo RNAs [including wt (oGJH151) or mt (oGJH152) ESS motif based on minigene mt9, and a control sequence (oGJH153) based on (mt1 and mt2)] were incubated with 293 or CAL 27 total cellular extract. Binding proteins were separated in SDS-PAGE gel and blotted with mouse anti-PTBP1 antibody. In panel A, the corresponding positions of oGJH151 and oGJH153 in exon 4 were labeled red and blue, respectively.
Figure 3
Figure 3. PTBP1 and PTBP2 inhibit the inclusion of SRSF3 exon 4.
(A–B) Overexpression of PTBP1 inhibited inclusion of exon 4. 293 cells were transfected with PTBP1 expression plasmid (A) or co-transfected with minigene and PTBP1 expression plasmid (B). Alternative splicing of exon 4 were analyzed by RT-PCR with primers located in the constant exon 3 and 7. An exon 3/5 forward junction primer was used to specifically amplify exon 4 excluded short product. Relative L/S represents the ratio of band intensities of long vs short isoforms. GAPDH served as loading control. Diagrams on the right of (A,B) show the structures of SRSF3 pre-mRNA and spliced products and primer positions (short lines above or below exons). (C) Overexpression of PTBP1 (fused with GFP) and T7 tagged SRSF3 were confirmed by western blot with specific anti-PTBP1 or SRSF3 antibody. (D) PTBP2 interacts with the ESS motif of SRSF3 exon 4. The western blot membranes in Fig. 2C was stripped and blotted with specific anti-PTBP2 antibody. (E–F) Knockdown of PTBP1 and PTBP2 promoted inclusion of exon 4. 293 cells were transfected by 20 nM PTBP1 (#1) and/or PTBP2, or non-specific (NS) siRNA. Alternative splicing of exon 4 were analyzed by RT-PCR with primers located in the constant exon 3 and 6 (E). Exon 4 specific and exon 3/5 forward junction primer were also used to specifically detect exon 4-included (L) and excluded (S) products, respectively (E). Western blot analysis showed the knockdown efficiency of PTBP1 and PTBP2 (F). Expression of SRSF3 is affected by knockdown of PTBP1 and PTBP2.
Figure 4
Figure 4. Co-expression and mutually regulation of SRSF3, PTBP1, and PTBP2 in OSCC cells.
(A) Expression of SRSF3, PTBP1, and PTBP2 in tumor and normal cells. Western blot membrane in Fig. 1A was further probed by specific anti-PTBP1 or PTBP2 antibody. T1 to T5 are primary oral squamous carcinoma cells. N1 to N3 are primary normal oral mucosal epithelial cell. (B) Overexpression of PTBP1 promoted SRSF3 expression in 293 or CAL 27 cells. Cells were transfected by PTBP1 (fused with GFP) expression plasmid or empty vector. (C) Knockdown of SRSF3 repressed the expression of PTBP1 and PTBP2 in 293 or CAL 27 cells. Cells were transfected by 20 nM SRSF3 siRNA or non-specific (NS) siRNA. The expression of PTBP1 and PTBP2 were analyzed by western blot. (D) Overexpression of SRSF3 promoted the expression of PTBP2. Western blot was used to analyze the expression of PTBP1 and PTBP2 in 293 cells transfected by T7 tagged SRSF3 expression plasmid or empty vector in Fig. 3C. β-actin served as loading control.
Figure 5
Figure 5. SRSF3 transcripts with exon 4 are NMD targets.
(A) CAL 27 or 293 cells were transfected with 20 nM UPF1 or non-specific (NS) siRNA twice in a 48-hour interval. Alternative splicing of SRSF3 exon 4 was analyzed by RT-PCR. Exon 4 specific and exon 3/5 forward junction primers were used to detect transcripts with exon 4 inclusion (L) and exclusion (S), respectively. Western blot analysis showed knockdown efficiency of UPF1. (B) NMD was inhibited indirectly by treating CAL 27 or 293 cells with 100 μg/ml CHX (Cell Signaling Technology, USA) for 5 h. Alternative splicing of SRSF3 exon 4 was analyzed by RT-PCR.
Figure 6
Figure 6. Model of SRSF3 expression regulated by PTBP1 and PTBP2 via controlling the alternative splicing of exon 4.
(A) The exon 4-excluded transcripts encode full length functional SRSF3. The exon 4-included transcripts were degraded via the NMD pathway, or may produce truncated SRSF3. In normal cells, SRSF3 regulates its own expression by inhibiting the exclusion of exon 4. (B) In cancer cells, overexpressed PTBP1 and PTBP2 bind to ESS motif and impair inclusion of exon 4, which then increases the expression of full length SRSF3 and promotes cell proliferation and transformation. Meantime, SRSF3, in turn, also promotes the expression of PTBP2.
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