The protein kinase Clk/Sty directly modulates SR protein activity: both hyper- and hypophosphorylation inhibit splicing

Abstract

The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

FIG. 1

SDS-PAGE analysis of recombinant proteins. Five milligrams each of Clk (lane 1), ClkR (lane 2), ecASF (lane 3), and bvASF (lane 4) were fractionated by SDS–10% PAGE and were stained with Coomassie blue R-250. The species indicated by the asterisk reflects an N-terminal truncation consisting of GST plus an approximate 10-kDa segment of Clk/Sty which becomes phosphorylated in the wild-type but not the mutant sample. The fragment is unlikely to have affected activity in our assays, as its presence was variable and not correlated with activity, and E. coli- and baculovirus-expressed Clks behaved similarly, although the latter lacks the truncated fragment (unpublished data). Lane M contains marker proteins (masses are indicated on the left in kilodaltons).

FIG. 2

Effects of Clk and ClkR on constitutive splicing. (A) Effect of Clk and ClkR on human β-globin pre-mRNA splicing in nuclear extracts. Lane 1, nuclear extract alone. Increasing amounts (50, 100, and 200 ng) of Clk (lanes 2 to 4) and ClkR (lanes 5 to 7) were added to nuclear extracts, and splicing was allowed to proceed for 90 min. RNAs were purified and fractionated by denaturing PAGE. (B) Effects of Clk and ClkR of β-globin splicing in S100 complemented with bvASF (300 ng). Lane 1, S100 plus bvASF only. Increasing amounts (25, 50, and 100 ng) of Clk (lanes 2 to 4) or ClkR (lanes 5 to 7) were added to reaction mixtures. The apparent loss of pre-mRNA in lanes 2 to 4 reflects poly(A) addition to the 3′ end of precursor, which resulted in upward smearing of the RNA during electrophoresis. (C) Effect of Clk and ClkR on β-globin splicing in S100 extracts complemented with ecASF (500 ng). Lane 1, S100 plus ecASF only; lanes 2 to 4 and 5 to 7, increasing amounts (50, 100, and 200 ng) of Clk and ClkR, respectively. The positions of the pre-mRNA, spliced product, and intermediates are depicted by symbols on the right.

FIG. 3

Both bvASF and ecASF can rescue Clk-mediated inhibition of constitutive splicing. (A) Effect of preincubation of Clk on bvASF and rescue of splicing. bvASF (200 ng) was preincubated (lanes 3 to 6) in the presence (lanes 4 to 6) or absence (lane 3) of 50 ng of Clk for 20 min in the absence of pre-mRNA. 50 or 100 ng of bvASF was added subsequently (lanes 5 and 6) along with the pre-mRNA (lanes 3 to 6), and splicing continued for 90 min. Samples in lanes 1 and 2 lacked or contained Clk, respectively, but were not preincubated. (B) Effect of preincubation of Clk on ecASF. ecASF (200 ng) was preincubated in the presence (lanes 2 to 8) or absence (lanes 1 and 9 to 12) of Clk. Subsequently, 50, 100, and 200 ng of ecASF (lanes 3 to 5 and 9 to 11) or GST-RS (lanes 6 to 8) were added along with pre-mRNA. Lane 12 contained 200 ng of GST-RS. The positions of the pre-mRNA, spliced product, and intermediates are indicated.

FIG. 4

Effects of Clk and ClkR on SR protein phosphorylation in splicing-competent extracts. (A) Western blot analysis of endogenous SR proteins in nuclear extracts. Aliquots of splicing reactions as in Fig. 2A were resolved by SDS-PAGE and subjected to Western blotting using MAb104. Lane 1, nuclear extract alone; lanes 2 to 4 and 5 to 7, increasing amounts of Clk and ClkR, respectively. The identities of specific SR proteins are indicated on the right, and masses (in kilodaltons) are indicated on the left. (B) Western blot analysis of S100 extracts containing ecASF. Aliquots of splicing reactions terminated at the indicated times were fractionated by SDS-PAGE and were analyzed by Western blotting as in panel A. Lanes 1 and 8, S100 extract alone; lanes 2, 5, and 9, S100 extracts plus 300 ng of ecASF; lanes 3, 6, and 10 and 4, 7, and 11, S100 plus 300 ng of ecASF and 100 ng of either Clk or ClkR, respectively. The positions of the three forms of ASF detected are indicated (see text). (C) Western blot analysis of S100 extracts containing bvASF. Conditions were exactly as in panel B, except that bvASF replaced ecASF and was present in all samples. Lanes 1 and 4 contain no added kinase, lanes 2 and 5 contain Clk, and lanes 3 and 6 contain ClkR. The positions of the two forms of ASF detected (see text) are indicated.

FIG. 5

Inhibition of ASF/SF2-activated human immunodeficiency virus tat splicing by Clk and ClkR. (A) Effect of Clk and ClkR on tat pre-mRNA splicing in S100 extracts complemented with ecASF (400 ng). Lane 1, ecASF alone; lanes 2 to 4 and 5 to 7, increasing amounts (50, 100, and 200 ng) of Clk and ClkR, respectively. (B) Effect of Clk and ClkR on S100 extracts complemented with bvASF (250 ng). Lane 1, bvASF alone; lanes 2 to 4 and 5 to 7, increasing amounts (25, 50, 100 ng) of Clk and ClkR, respectively. (C) Effect of ClkR on ecASF-mediated activation of splicing in the presence of SC35. Lanes 1 and 3 to 5, 100 ng of ecASF; lanes 2 to 5, 200 ng of SC35; lanes 4 and 5, 100 and 200 ng of ClkR, respectively. The positions of the pre-mRNA, spliced product, and intermediates are shown. ∗ indicates an artifactual cleavage product unrelated to splicing.

FIG. 6

Clk and ClkR modulate SV40 pre-mRNA alternative splicing. (A) Effect of Clk and ClkR on SV40 pre-mRNA splicing in nuclear extract. Lane 1, nuclear extract alone; lanes 2 to 4 and 5 to 7, increasing amounts (50, 100, and 200 ng) of Clk and ClkR, respectively. (B) Effect of Clk and ClkR on SV40 pre-mRNA splicing in S100 extracts complemented with ecASF (750 ng). Lane 1, ecASF only; lanes 2 to 4 and 5 to 7, increasing amounts (50, 100, and 200 ng) of Clk and ClkR, respectively. Positions of pre-mRNA, spliced products, and intermediates are shown. A schematic of the pre-mRNA is shown at the bottom.

FIG. 7

Clk and ClkR modulate adenovirus E1a pre-mRNA alternative splicing in S100 extracts complemented with SC35. All samples contained E1a pre-mRNA and baculovirus-produced SC35 (500 ng). Lane 1, SC35 only; lanes 2 and 3 and 4 and 5, increasing amounts (100 and 200 ng) of Clk and ClkR, respectively. Positions of pre-mRNA, spliced products, and intermediates are shown. A schematic of the pre-mRNA is shown at the bottom.