PSKH1, a novel splice factor compartment-associated serine kinase

Abstract

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in splicing factor compartments (SFCs) within the nucleus of interphase cells. Nuclear SFCs are considered mainly as storage sites for splicing factors, supplying splicing factors to active genes. The mechanisms controlling the interaction of the various spliceosome constituents, and the dynamic nature of the SFCs, are still poorly understood. We show here that endogenous PSKH1, a previously cloned kinase, is located in SFCs. Migration of PSKH1-FLAG into SFCs is enhanced during co-expression of T7-tagged ASF/SF2 as well as other members of the SR protein family, but not by two other non-SR nuclear proteins serving as controls. Similar to the SR protein kinase family, overexpression of PSKH1 led to reorganization of co-expressed T7-SC35 and T7-ASF/SF2 into a more diffuse nuclear pattern. This redistribution was not dependent on PSKH1 kinase activity. Different from the SR protein kinases, the SFC-associating features of PSKH1 were located within its catalytic kinase domain and within its C-terminus. Although no direct interaction was observed between PSKH1 and any of the SR proteins tested in pull-down or yeast two-hybrid assays, forced expression of PSKH1-FLAG was shown to stimulate distal splicing of an E1A minigene in HeLa cells. Moreover, a GST-ASF/SF2 fusion was not phosphorylated by PSKH1, suggesting an indirect mechanism of action on SR proteins. Our data suggest a mutual relationship between PSKH1 and SR proteins, as they are able to target PSKH1 into SFCs, while forced PSKH1 expression modulates nuclear dynamics and the function of co-expressed splicing factors.

Figure 1

PSKH1 localizes to SFCs. Endogenous PSKH1 partly localizes to nuclear speckles in U2OS cells (A). Endogenous SC35 as a SFC marker (B). When superimposed, PSKH1 and SC35 at least partly co-localize to SFCs (C). PSKH1-FLAG (D). Endogenous SC35 (E). PSKH1-FLAG and endogenous SC35 superimposed (F). U2OS cells were transfected with PSKH1-FLAG (G, J and M) and co-transfected with a 4-fold molar excess of one of the following T7-tagged SR protein expression plasmids: T7-ASF/SF2 (H), T7-SC35 (K), T7-9G8 (N). (G), (J) and (M) and (H), (K) and (N) are superimposed in (I), (L) and (O), respectively. Endogenous PSKH1 was detected by a rabbit anti-PSKH1 antibody and a FITC-conjugated swine anti-rabbit antibody, while endogenous SC35 was detected by a monoclonal anti-SC35 antibody and a Texas red-conjugated donkey anti-mouse secondary antibody. The double transfected cells were labeled with anti-PSKH1 and anti-T7 antibodies using the same secondary antibodies as for endogenous staining. Scale bar = 10 µM.

Figure 2

PSKH1 targets SFCs through its kinase core and C-terminal domains. Three PSKH1 deletion mutants: Δ95-424 (1-94-EGFP), Δ1-77, Δ361-424 (FLAG-78-360) and Δ1-354 (355-424-EGFP) were separately co-transfected into U2OS cells with a 4-fold molar excess of T7-ASF/SF2 or T7-SC35 expression plasmid. 1-94-EGFP in cells co-expressing T7-ASF/SF2 (A). Same cell as in (A) visualizing T7-ASF/SF2 (B). (A) and (B) are superimposed in (C). The catalytic kinase domain (FLAG-78-360) migrates into SFCs when T7-ASF/SF2 is co-expressed (D). T7-ASF/SF2 speckles during FLAG-78-360 co-expression (E). (D) and (E) are superimposed in (F). The C-terminal EGFP-355-424 fusion (G) shows no significant increase in SFCs when co-expressed with T7-ASF/SF2 (H). EGFP-355-424 and T7-ASF/SF2 are superimposed in (I). EGFP-355-424 migrates into SFCs when co-expressed with T7-SC35 (J). T7-SC35 in cells co-expressing EGFP-355-424 (K). EGFP-355-424 and T7-SC35 are superimposed in (L). A reduced size frame (phase contrast) of the same cell demonstrates the dense nucleolus structures (L, inset; arrowheads indicate increased staining of nucleolar structures). Control of the intracellular localization pattern of 1-94-EGFP without co-expressing T7-ASF/SF2 (M) (nucleus stained blue), FLAG-78-360 without co-expressing T7-ASF/SF2 (N) and EGFP-355-424 without co-expressing T7-ASF/SF2 (O). Scale bar = 10 µM.

Figure 3

(A) Protein expression level of PSKH1-FLAG and T7-SC35 in co-transfection assays. Immunoblot analysis of cells co-transfected with a 4-fold molar excess of T7-SC35 relative to PSKH1-FLAG (lane 1) and a 4-fold molar excess of PSKH1-FLAG relative to T7-SC35 (lane 2). The relative level of each co-expressed tagged protein is shown at the bottom of the figure. (B) Transient overexpression of PSKH1-FLAG leads to a redistribution of co-expressed T7-SC35 and T7-ASF/SF2. U2OS cells were co-transfected with PSKH1-FLAG (A–F) or its kinase-negative mutant (G–I) and T7-ASF/SF2 (A–C) or T7-SC35 (D–I). A 4-fold molar excess of PSKH1 coding plasmid (or its corresponding mutant DNA) compared to SR coding plasmid was applied. Forced expression of PSKH1-FLAG (or mutant) antagonizes the normal speckled appearance of SC35 (E, F, H and I, small arrowheads) in more than 70% of the double transfected cells. Cells not expressing PSKH1 (or mutant) (large arrowheads) all show a normal speckled pattern. The localization of PSKH1-FLAG in the Golgi apparatus was often strongly reduced or absent in cells co-expressing T7-tagged SR protein (D and F). Scale bar = 10 µM.

Figure 4

Nuclear reorganization of PSKH1 during transcriptional arrest. Endogenous PSKH1 (A) or transfected EGFP-PSKH1 fusion (D) reorganizes into enlarged and rounded-up speckles together with endogenous SC35 (B and E) upon transcriptional arrest (C and F). Scale bar = 10 µM.

Figure 5

PSKH1 affects splicing of adenovirus E1A pre-mRNA in HeLa cells. (A) Schematic representation of the E1A minigene, showing various alternative splice sites, the positions of the primers used for RT–PCR and the expected lengths of the resulting RT–PCR products. (B) Lane 1, mRNA expressed from the plasmid pMTE1A co-transfected with pT7-ASF/SF2; lane 2, plasmid pMTE1A co-transfected with the vector control; lanes 3–5, plasmid pMTE1A co-transfected with increasing amounts of pPSKH1-FLAG (0.05, 0.5 and 1.5 µg); lane 6, MassRuler™ DNA ladder, low range (Fermentas). Each bar represents the average of three experiments and shows schematically the level of each splice product. As controls for PSKH1-FLAG protein expression, identical plasmid combinations as used in the RT–PCR analysis were run in parallel and subjected to immunoblot analysis. PSKH1-FLAG was detected using a monoclonal anti-FLAG antibody (M2). As a control for equal loading, β-tubulin was monitored with an anti-β-tubulin antibody. Protein expression level control for T7-ASF/SF2 was performed using the anti-T7 antibody. (C) Lane 1, MassRuler™ DNA ladder, low range (Fermentas); lanes 2–4, plasmid pMTE1A co- transfected with increasing amounts of pPSKH1-D218A-FLAG (0.05, 0.5 and 1.5 µg). Each bar represents the average of two experiments. As a control for PSKH1_D218A-FLAG protein expression, parallels with identical plasmid combinations (as used in the RT–PCR assay for lanes 2–4) were transfected into cells and subjected to immunoblot analysis. β-Tubulin was used as a control for equal loading. (D) Lane 1, MspI-digested pBR322; lane 2, plasmid pMTE1A co-transfected with pFLAG-78-360 (1.5 µg); lane 3, plasmid pMTE1A co-transfected with pT7-ASF/SF2 (0.5 µg); lane 4, plasmid pMTE1A co-transfected with pFLAG-78-424 (1.5 µg); lane 5, plasmid pMTE1A co-transfected with vector control (1.5 µg). The results in (D) are representative of at least three separate experiments for each expression plasmid. The positions of the 13S, 12S and 9S transcripts are indicated at the side of each gel. Gels were stained with Cyber Gold. RNA was harvested 24 h after transfection and analyzed by RT–PCR. DNA standard, MspI-digested pBR322.