Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae

Abstract

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.

Figure 1

Sky1p phosphorylates a single site in Npl3p. (a) Sequence of the RGG/RS domain in Npl3p. RGG repeats and SR/RS dipeptides are underlined and in bold, respectively. Deletion mutants are named according to the number of SR/RS dipeptides removed, and specific point mutations in the deduced phosphorylation site are indicated. (b) Phosphorylation consensus deduced from in vitro peptide selection using human SRPK2 (Wang et al. 1998). (c) Wild-type and mutant Npl3p were expressed as GST fusion proteins (Coomassie stain, top) and tested for phosphorylation with recombinant Sky1p (Autoradiograph, bottom).

Figure 2

Defects in Sky1p-mediated phosphorylation result in Npl3p accumulation in the cytoplasm. (a) All GFP fusion proteins were intact as determined by Western blotting analysis using an anti-GFP mAb. (b) Localization of wild-type (wt) and mutant GFP-npl3p in wild-type (SKY1) and sky1Δ strains.

Figure 3

Sky1p-mediated phosphorylation plays an important role in facilitating Npl3p–Mtr10p interaction. (a) Coimmunoprecipitation between Npl3p and Mtr10p. Immunoprecipitation (IP) was done with IgG-Sepharose beads and analyzed by Western blotting using rabbit polyclonal anti-Npl3p antibodies and IgG to detect Npl3p and Mtr10-PrA, respectively. Quantification of these coimmunoprecipitation results revealed a threefold reduction of Npl3p binding to Mtr10p-PrA in sky1Δ yeast. (b) In vitro GST binding assay. Wild-type and mutant (S411A) GST-npl3p were phosphorylated (+P) using Sky1p or mock phosphorylated (−P), and used to pull down in vitro–translated ³⁵S-labeled Mtr10p in the absence (lanes 2–5) or presence (lanes 6–9) of cytosolic yeast extract. (c) In vitro binding using purified components. The binding was done as in b, except that equal amounts (1 μg) of purified recombinant GST-Npl3p and His-S-Mtr10p were used. Bound His-S-Mtr10p was detected with alkaline phosphatase–conjugated S-protein.

Figure 3

Sky1p-mediated phosphorylation plays an important role in facilitating Npl3p–Mtr10p interaction. (a) Coimmunoprecipitation between Npl3p and Mtr10p. Immunoprecipitation (IP) was done with IgG-Sepharose beads and analyzed by Western blotting using rabbit polyclonal anti-Npl3p antibodies and IgG to detect Npl3p and Mtr10-PrA, respectively. Quantification of these coimmunoprecipitation results revealed a threefold reduction of Npl3p binding to Mtr10p-PrA in sky1Δ yeast. (b) In vitro GST binding assay. Wild-type and mutant (S411A) GST-npl3p were phosphorylated (+P) using Sky1p or mock phosphorylated (−P), and used to pull down in vitro–translated ³⁵S-labeled Mtr10p in the absence (lanes 2–5) or presence (lanes 6–9) of cytosolic yeast extract. (c) In vitro binding using purified components. The binding was done as in b, except that equal amounts (1 μg) of purified recombinant GST-Npl3p and His-S-Mtr10p were used. Bound His-S-Mtr10p was detected with alkaline phosphatase–conjugated S-protein.

Figure 5

SKY1 and MTR10 show a genetic interaction. (a) Overexpression suppression of Npl3p localization defect. The left panels show localization of mutant GFP-npl3p(S411A) in wild-type yeast containing empty vector (p415) or a GAL-inducible plasmid overexpressing Mtr10p (p415-MTR10). The right panel shows a similar analysis on localization of wild-type GFP-Npl3p in sky1Δ yeast. (b) Synthetic lethality between SKY1 and MTR10. A sky1Δ strain was first covered with a Ura⁺ plasmid containing SKY1 (pRS316-SKY1). Transformants with (top left) or without (bottom right) targeted disruption of MTR10 were streaked on a 5-FOA plate and incubated at 30°C. SKY1 (bottom left) or MTR10 (top right) deletion strains were used as controls.

Figure 4

Mtr10p interacts with the RGG box in Npl3p. (a) Deletion mapping of the Mtr10p interaction domain in Npl3p. Binding was done as in the legend to Fig. 3 b using deletion mutants illustrated in Fig. 1 a. (b) Plasmid shuffling experiments. An npl3Δ strain covered by wild-type NPL3 (wt) on a Ura⁺ plasmid was transformed with Leu⁺ plasmids containing wild-type or mutant npl3. Transformants were streaked on 5-FOA minus Leu plates and incubated at 25 or 37°C.

Figure 6

In vivo evidence for the negative regulation of Npl3p nuclear import by the arginine methyltransferase Hmt1p/Rmt1p. (a) Localization of GFP-Npl3p in wild-type yeast transformed with empty vector (p415) or with Hmt1p/Rmt1p overexpression plasmid (labeled p415-HMT1). (b) Immunoblot analysis of HA-tagged Hmt1p/Rmt1p expression before and after galactose induction. The asterisk indicates a cross-reactive band in yeast extract against the anti-HA antibody. (c) Hypermethylation of Npl3p. Npl3p methylation was detected by the methylation specific mAb 1E4 and total Npl3p by rabbit polyclonal anti-Npl3p. (d) Coimmunoprecipitation between Npl3p and Mtr10p-PrA was done as in the legend to Fig. 3 a. Complex formation was compared among wild-type, sky1Δ, and Hmt1p/Rmt1p overexpressing cells.

Figure 7

Arginine methylation interferes with phosphorylation-dependent binding through an indirect mechanism. (a) Methylation of wild-type (wt) and mutant npl3p using purified Hmt1p/Rmt1p in the presence of the methyl donor ³H-SAM. (b) In vitro GST binding assay. GST-Npl3p methylated (+M) or mock methylated (−M) were used to pull down in vitro–translated ³⁵S-labeled Mtr10p in the absence (lanes 2–4) or presence (lanes 5–7) of yeast extract. (c) Effect of double modification on Npl3p binding to Mtr10p. GST-Npl3p was either phosphorylated using Sky1p (lanes 4 and 7) or mock phosphorylated (lanes 3 and 6) and then methylated using Hmt1p/Rmt1p. Conversely, the fusion protein was mock methylated (lane 8) or methylated (lane 9) and then phosphorylated. These modified proteins were used in the GST binding assay as in b.

Figure 8

Arginine methylation antagonizes Sky1p-mediated phosphorylation. (a) Constructs for in vitro phosphorylation and methylation. The single Sky1p phosphorylation site at the COOH-terminal region of Npl3p and multiple Hmt1p/Rmt1p methylation sites in the RGG domain are indicated. Iκb/RS represents a fusion protein containing Iκb and the Sky1p phosphorylation site from Npl3p. (b) Methylation of phosphorylated or mock-phosphorylated wild-type Npl3p and deletion mutant Δ1, indicating that phosphorylation does not affect methylation. (c) The proteins illustrated in d were methylated using Hmt1p/Rmt1p or mock methylated, and then tested for phosphorylation using Sky1p. (d) Phosphorylation of Npl3p that was untreated, mock methylated in the presence of SAH, or methylated with SAM.

Figure 9

A model for Npl3p nuclear import and export controlled by Sky1p-mediated phosphorylation and Hmt1p-mediated methylation. (a) In wild-type cells, Sky1p in the cytoplasm phosphorylates Npl3p, which is required for its efficient nuclear import by Mtr10p. Imported Npl3p is methylated by Hmt1p/Rmt1p in the nucleus, which may faciliate Npl3p export. Methylation of Npl3p interferes with Sky1p-mediated phosphorylation, thereby indirectly inhibiting Npl3p import. The size of the arrows indicates relative transport efficiency, and shaded regions indicate Npl3p localization at steady state. (b) Impaired nuclear import of wild-type Npl3p in the nucleoporin mutant nup49–313 strain (based on data from Lee et al. 1996). (c) Impaired nuclear import of the phosphorylation mutant npl3pE409K in wild-type yeast. (d) Deletion of HMT1/RMT1 improves import and impairs export of Npl3p in the nup49–313 strain. (e) Import and export are affected by Npl3p mutation in the phosphorylation site and inactivation of Hmt1p/Rmt1p, respectively (our observation, and that reported by McBride et al. 2000).