Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Clk1 enhances SPF45-induced exon 6 exclusion from Fas mRNA. ( A ) SKOV3 cells were transfected with siRNA against SPF45 (siSPF45) or scrambled control siRNA (scr) for 72 h, and RNA was isolated. Endogenous Fas spliced isoforms were analysed by RT-PCR using primers flanking exon 6. PCR products representing mRNA including exon 6 [Fas (L)] and excluding exon 6 [Fas (S)] are shown and are quantified in the graph, with the ratio Fas (S) to Fas (L) set to one in the control siRNA group. The resuslts are from three independent experiments performed in duplicate and were statistically significant. ( B ) Parallel cultures to those in (A) were lysed for western blotting using antibodies to SPF45 and actin. ( C ) Cell lysates from the indicated cell lines were immunoblotted for SPF45 using a polyclonal antibody to recombinant His-SPF45. COS-1 cells transfected with untagged SPF45 (COS-1, +con) and OV2008 cells expressing control or SPF45-specific shRNA served as positive controls. The SPF45 band (SPF45) and an SPF45 degradation product (degr.) in the positive control lane are labelled. ( D ) COS-1 cells were cotransfected with Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) and endogenous Fas spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratio of exon 6 exclusion from three experiments done in duplicate are shown under the gel images. ( E ) Schematic of the Δ Fas minigene splicing reporter used in transfection assays and the different mRNA isoforms derived from it, representing inclusion or exclusion of exon 6. ( F ) SPF45 expression induced exon 6 exclusion in a dose-dependent manner. COS-1 cells were co-transfected with Δ Fas and increasing amounts of Myc-SPF45. Total RNA was extracted at 24 h and analysed by RT-PCR using Δ Fas -specific primers. A representaive gel from at least three independent experiments is shown, and the ratio of the lower band to the upper band is shown below the gel. The lower panel represents a western blot of Myc-SPF45 and actin protein expression from one experiment. ( G ) Clk1 overexpression promotes SPF45 alternative splicing activity. COS-1 cells were cotransfected with Δ Fas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratios of exon 6 exclusion are shown under the gel images. Results were derived from three independent experiments done in duplicate. Statistical significance ( P < 0.01) is indicated in the graph. ( H ) Clk1 overexpression enhanced SPF45 protein levels. Protein lysates were prepared from cells transfected as in (G) and were subjected to western blotting using antibodies to Myc, Clk1 and actin.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Transfection, Control, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Recombinant, Expressing, shRNA, Positive Control, Derivative Assay, Over Expression, Alternative Splicing, Activity Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Inhibition of Clk1 decreases both SPF45 protein levels and exon 6 exclusion by SPF45. ( A ) COS-1 cells were cotransfected with Δ Fas (0.3 μg), Myc-SPF45 (0.6 μg) and Clk1 (0.8 μg) or Clk1-K233R (0.8 μg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The graph under the gel image represents the quantification of the corresponding bands from three experiments done in duplicate. ( B ) COS-1 cells were cotransfected as in (A), and whole-cell protein lysates were immunoblotted with anti-Myc and anti-actin antibodies. ( C ) COS-1 cells were pretreated with the Clk inhibitor TG003 (10 μM) for 1 h and then cotransfected with Δ Fas and Myc-SPF45 or empty vector. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. A graph under the gel image showed the relative ratio of the lower band to the upper band. The results were derived from three independent experiments done in duplicate and statistical significance ( P < 0.01) is indicated. ( D ) COS-1 cells were pretreated with TG003 for 1 h and then cotransfected as in (C). Whole-cell protein lysates were immunoblotted as in (B).

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Knockdown of Clk1 decreases both SPF45 protein levels and SPF45-induced exon 6 exclusion. ( A ) COS-1 cells were transfected with three siRNA against Clk1 or scrambled control siRNA (scr). Total RNA was extracted at 48 h post-transfection and was subjected to real-time PCR analysis using primers specific to Clk1. ( B ) COS-1 cells were transfected as in (A), and whole-cell protein lysates were immunoblotted with anti-Clk1 and anti-actin antibodies. ( C ) COS-1 cells were transfected with siRNA against Clk1 (siClk1-1, siClk1-2 and siClk1-3) or scrambled control siRNA for 48 h and then cotransfected with plasmids for ΔFas and SPF45 or empty vector. Twenty-four hour after plasmid transfection, mRNA was collected, and Δ Fas spliced products were analysed by RT-PCR. ( D ) Graph showing the relative ratio of splicing products (the lower band to the upper band) after quantification of the corresponding lanes in panel (C). The ratio of splicing products for the scrambled siRNA in the presence of transfected SPF45 was set to 1. The results are from three independent experiments done in duplicate and statistical significance ( P < 0.01) is indicated. ( E ) COS-1 cells were transfected as in (C), and whole-cell protein lysates were immunoblotted with anti-Myc and anti-actin antibodies.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Knockdown, Transfection, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Clk1 inhibition decreases the half-life of SPF45 through a proteasome-dependent pathway. ( A ) A2780 cells were treated with cycloheximide (50 μg/ml) and TG003 (10 μM) or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies to detect endogenous proteins. ( B ) Graph of SPF45 protein levels relative to the actin loading control from (A). ( C ) SKOV-3-Myc-SPF45 stable cells were treated with cycloheximide and TG003 or DMSO for the indicated times followed by western blot analysis with anti-Myc and anti-actin antibodies. ( D ) Graph of Myc-SPF45 protein levels relative to actin from (C). ( E ) Knockdown of Clk1 inhibits expression of endogenous SPF45. SKOV-3 cells and HeLa cells were transfected with siRNA to Clk1 or control siRNA (scr) for 48 h. Cell lysates were immunoblotted for endogenous Clk1, SPF45 and actin. ( F ) The increased degradation of SPF45 with Clk1 inhibition is proteasome-dependent. A2780 cells were treated with MG132 (10 μM) and TG003 or DMSO for the indicated times, followed by western blot analysis with anti-SPF45 and anti-actin antibodies. Quantitation of SFP45 expression relative to actin is below each lane, with the DMSO control lane set to 1.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Inhibition, Western Blot, Control, Knockdown, Expressing, Transfection, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Clk1 phosphorylates eight serines in SPF45. ( A ) His-SPF45 was incubated in the absence or presence of recombinant CLK1 and [γ-32 P]-ATP in an in vitro kinase assay. The reactions were run on a gel, transferred to nitrocellulose and exposed for autoradiography, followed by western blotting for SPF45. ( B ) Recombinant Clk1 was used to phosphorylate His-SPF45 from bacteria in vitro , and the phosphorylated protein was run on a gel and processed for mass spectrometry. Eight phosphorylated serine residues in SPF45 were identified. ( C ) Recombinant Clk1 was used to phosphorylate His-SPF45, His-SPF45-S202A, His-SPF45-S204A, His-SPF45-6 A (S48/62/222/266/288/291 A) and His-SPF45-8 A (S48/62/202/204/222/266/288/291 A) in vitro using [γ- 32 P]-ATP. The reactions were run on a gel, transferred to nitrocellulose, exposed for autoradiography and immunoblotted with anti-SPF45 antibody. ( D ) COS-1 cells were transfected with empty vector, Myc-SPF45 or Myc-SPF45-8 A. Anti-Myc immunoprecipitates were immunoblotted with anti-phospho-serine and anti-Myc antibodies.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Incubation, Recombinant, In Vitro, Kinase Assay, Autoradiography, Western Blot, Bacteria, Mass Spectrometry, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Mutation of Clk1 phosphorylation sites on SPF45 regulates SPF45 splice site utilization. ( A ) COS-1 cells were transfected for Δ Fas splicing assays as above using either Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D in the absence or presence of either empty vector or Clk1. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. A representative gel is shown. ( B ) The means and SE for the relative ratios of exon 6 exclusion from (A) are shown in the graph. Results were derived from three independent experiments in duplicate and statistical significance ( P < 0.01) is indicated. ( C ) The same as in (A), but total RNA was subjected to RT-PCR analysis using primers specific to Myc-SPF45 and GAPDH. ( D ) Protein lysates from cells transfected in parallel were immunoblotted with anti-Myc and anti-actin antibodies.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Mutation of Clk1 phosphorylation sites in SPF45 differentially affects SPF45-indcuced exon 6 exclusion . ( A and B ) COS-1 cells were transfected with plasmids for Δ Fas (0.3 μg), wild-type (wt)-SPF45 (0.6 μg) or an SPF45 mutant (0.6 μg) as indicated. Twenty-four hour post-transfection, spliced products were analysed by RT-PCR. The graph shows the relative ratios (short form to long form) of exon 6 exclusion. The table under the graph indicates the means and SE for each SPF45 mutant. Results were derived from three independent experiments done in duplicate. * P < 0.05 versus wt group, ** P < 0.01 versus wt group. The bottom panels show western blotting of protein lyasates using anti-Myc and anti-actin antibodies.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Mutation of the Clk1 phosphorylation sites in SPF45 affects Δ Fas mRNA binding, but not binding to other splicing factors. ( A ) COS-1 cells were transfected with either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. After 24 h, the cells were lysed, and anti-Myc immunoprecipitates were run on a gel and immunoblotted for Myc and co-immunoprecipitating endogenous SF3b155, U2AF65 and SF1. Cell lysates (Lys.) were immunoblotted as indicated. ( B ) COS-1 cells were co-transfected with Δ Fas and either empty vector, Myc-SPF45, Myc-SPF45-8 A or Myc-SPF45-8D. Twenty-four hour after transfection, the cells were subjected to RNA IP using anti-Myc antibody followed by RT-qPCR analysis to detect the binding of Δ Fas mRNA to Myc-SPF45. Relative binding to the control IP from six experiments are shown as mean ± SE. * P < 0.05 versus vector group, # P < 0.05 versus wt-SPF45 group.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Mutagenesis, Phospho-proteomics, Binding Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Control

Journal: Nucleic Acids Research

Article Title: Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

doi: 10.1093/nar/gkt170

Figure Lengend Snippet: Clk1 regulates SPF45-induced exon 6 exclusion from fas mRNA through antagonistic mechanisms. The model shows that Clk1 regulates SPF45 splice site utilization through multiple mechanisms involving increases in SPF45 stability, phosphorylation and regulation of mRNA binding. SPF45 overexpression causes enhanced migration and invasion, dependent on the identified phosphorylation sites and regulation of fibronectin and cortactin.

Article Snippet: Quantitative real-time PCR was carried out using either Taqman Gene Expression Assays: Hs00269734_m1 for Clk1 and Hs99999901_sl for the housekeeping gene of 18S RNA on a 7300 Real-Time PCR System (Applied Biosystems) or Bio-Rad’s SsoAdvancedTM SYBR® Green Supermix on an Eppendorf (Hauppage, NY) Mastercycler Realplex 2.

Techniques: Phospho-proteomics, Binding Assay, Over Expression, Migration