Arginine methylation of the nuclear poly(a) binding protein weakens the interaction with its nuclear import receptor, transportin

Abstract

The nuclear poly(A) binding protein, PABPN1, promotes mRNA polyadenylation in the cell nucleus by increasing the processivity of poly(A) polymerase and contributing to poly(A) tail length control. In its C-terminal domain, the protein carries 13 arginine residues that are all asymmetrically dimethylated. The function of this modification in PABPN1 has been unknown. Part of the methylated domain serves as nuclear localization signal, binding the import receptor transportin. Here we report that arginine methylation weakens the affinity of PABPN1 for transportin. Recombinant, unmethylated PABPN1 binds more strongly to transportin than its methylated counterpart from mammalian tissue, and in vitro methylation reduces the affinity. Transportin and RNA compete for binding to PABPN1. Methylation favors RNA binding. Transportin also inhibits in vitro methylation of the protein. Finally, a peptide corresponding to the nuclear localization signal of PABPN1 competes with transportin-dependent nuclear import of the protein in a permeabilized cell assay and does so less efficiently when it is methylated. We hypothesize that transportin binding might delay methylation of PABPN1 until after nuclear import. In the nucleus, arginine methylation may favor the transition of PABPN1 to the competing ligand RNA and serve to reduce the risk of the protein being reexported to the cytoplasm by transportin.

FIGURE 1.

Arginine methylation of PABPN1 reduces the affinity for transportin. A, amino acid sequence of the C-terminal domain of bovine PABPN1. Dimethylated arginine residues are underlined. Amino acids conforming to the transportin binding site consensus are boxed. B, recombinant non-methylated His-tagged and non-tagged PABPN1 (His tag, no tag), fully methylated PABPN1 from calf thymus (c.t.), and BSA were covalently immobilized on NHS-activated Sepharose beads. Each immobilized protein (20 μg, approximately 2.5 μm for PABPN1) was incubated with HeLa cell nuclear extract (2.3 mg of protein) for 4 h at 8 °C (volume, 250 μl). Beads were washed, and bound proteins were eluted with SDS. 25% of each elution fraction was analyzed by SDS-PAGE and immunoblotting with antibodies against transportin. Size markers (in kDa) are indicated on the right. The membrane used here was the same as in Fig. 6 of Ref. . By probing the membrane for PABPN1, we showed in that work that PABPN1 present in the extract associated with all three variants of the immobilized protein. This serves as a control that all beads had been loaded with protein and that similar amounts of extract were applied.

FIGURE 2.

Recombinant transportin binds specifically to PABPN1 and prefers unmethylated PABPN1. Recombinant non-methylated His-tagged and non-tagged PABPN1 (his tag, no tag), fully methylated PABPN1 from calf thymus (c.t.), the recombinant non-methylated deletion variant PABPN1-ΔC8 (ΔC8), and BSA were covalently immobilized on NHS-activated Sepharose beads. Each immobilized protein (5 μg, 0.3 μm for PABPN1) was incubated with recombinant transportin 1 (6 μg, 0.12 μm) in 500 μl of binding buffer A containing 350 mm KCl, 5 mm MgCl₂ and 1.25 mg/ml BSA for 2 h at 8 °C. The salt concentration used corresponds to that introduced by the non-dialyzed high-salt nuclear extract used in Fig. 1. Where indicated, RanQ69L loaded with GTP (0.5 μm) was added to the reactions. Beads were washed, and bound proteins were eluted with SDS and analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie staining.

FIGURE 3.

Transportin binding of PABPN1 is sensitive to in vitro methylation. Recombinant PABPN1 was methylated in vitro (+ SAM) or mock-methylated (- SAM). For each time point, the reaction contained 1 μg His-PRMT1, 1 μg His-PABPN1 and, when present, 6 nmol of S-adenosyl-L-methionine in a volume of 25 μl. An additional reaction was carried out with 5 nmol of S-adenosyl-l-methionine and 1 nmol [¹⁴C]S-adenosyl-L-methionine under otherwise identical conditions. Reactions were stopped after incubation for 0, 15, 30, 60, or 120 min by the addition of 15 nmol of S-adenosyl-L-homocysteine and freezing in liquid nitrogen. The reaction containing the ¹⁴C-labeled methyl group donor was used for quantification of methylation. The extent of methylation is indicated at the top. 100% methylation corresponds to 26 methyl groups incorporated per PABPN1 molecule. For binding experiments, immobilized transportin1 (2 μg, 0.1 μm) was incubated with PABPN1 (0.3 μg, 0.05 μm) from the various time points of the “cold” methylation reaction (left) or from the mock reaction (right). Binding reactions were carried out as in Fig. 2 in a volume of 200 μl. Beads were washed, and bound proteins were eluted with SDS and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with the single chain antibody 3F5 (upper panel). Equal input of all binding experiments was analyzed by immunoblotting with the same antibody (lower panel).

FIGURE 4.

Interaction between PABPN1 and transportin is competed by the NLS of PABPN1 in a methylation-dependent manner. Immobilized PABPN1 or BSA (2.5 μg, 0.4 μm for PABPN1) were incubated with recombinant transportin 1 (5 μg, 0.25 μm, 50 pmol) and increasing amounts of methylated (□) or unmethylated (●) PABPN1-NLS peptides. Binding reactions were carried out for 1 h at room temperature in binding buffer A with 150 mm KCl, 5 mm MgCl₂, and 1.25 mg/ml BSA in a volume of 200 μl. The molar excess of peptide over immobilized PABPN1 is indicated at the top. Beads were washed and bound proteins were eluted with SDS and analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining and scanning. A, Coomassie staining. B, data from two independent experiments are combined.

FIGURE 5.

Transportin competes with RNA binding and methylation of PABPN1. A, 10 fmol (0.25 nm) radiolabeled A₁₄ and 50 nm of unmethylated (from E. coli, ●) or methylated PABPN1 (from calf thymus, □) were preincubated for 15 min at room temperature. Transportin 1 (0–4 μm) was added, and the reactions were incubated for an additional 20 min. Filter binding assays were carried out as described under “Experimental Procedures,” and RNA retained on the filter was plotted against transportin concentration. RNA binding in the absence of PABPN1 was subtracted as background. This was 0.1 fmol when both PABPN1 and transportin were omitted and increased to 0.6 fmol at 1 μm and 1.45 fmol at 4 μm transportin. B, recombinant PABPN1 (2 pmol, 100 nm) was methylated with His-PRMT1 (14 nm) for 30 min at 30 °C with [¹⁴C]S-adenosylmethionine as the methyl donor. Transportin was present as indicated. Proteins were separated by SDS-PAGE, and in vitro methylation was quantified by autoradiography. Reactions were carried out in the absence (◊) or in the presence (▾) of 4 μm RanQ69L loaded with GTP.

FIGURE 6.

Nuclear import of PABPN1 is mediated by transportin. A, 0.5 μm FITC-labeled PABPN1 or PABPN1ΔC25 were preincubated for 10–12 min under import conditions as described under “Experimental Procedures” with or without 0.75 μm transportin. Import reaction mixtures were then incubated with permeabilized HeLa cells for 18 min at room temperature. Cells were fixed with 4% paraformaldehyde, and import was analyzed by fluorescence microscopy. B, 0.5 μm FITC-labeled PABPN1 or Texas Red-labeled BSA-NLS were preincubated for 10–12 min under import conditions as described under “Experimental Procedures” with or without 0.75 μm importin α and 0.5 μm importin β. Import reaction mixtures were then incubated with permeabilized HeLa-cells for 18 min (FITC-PABPN1) or 6 min (BSA-NLS) at room temperature. Cells were fixed with 4% paraformaldehyde, and import was analyzed by fluorescence microscopy.

FIGURE 7.

Nuclear import of PABPN1 is efficiently competed by the unmethylated PABPN1-NLS. A, 0.5 μm FITC-labeled PABPN1 was preincubated for 10–12 min under import conditions with or without 0.3 μm transportin as described under “Experimental Procedures.” Import reaction mixtures were then incubated with permeabilized HeLa-cells for 18 min at room temperature. Cells were fixed with 4% paraformaldehyde, stained with DAPI, and import was analyzed by confocal microscopy. B, the reactions as in A were carried out in the presence of 0.2 or 1 μm methylated (left panel) or unmethylated (right panel) NLS peptide. C, the import of PABPN1 for each experiment was quantified for 70 cells as described under “Experimental Procedures.”

FIGURE 8.

PRMT1 knockout increases binding of cellular FUS/TLS to transportin. Nuclear extracts of ES wild-type (ES +/+) or ES-PRMT1 knockout (ES −/−) cells were supplemented with zz-transportin (+) and incubated with IgG-Sepharose beads as described under “Experimental Procedures.” Bound proteins were washed, eluted by GTP-loaded RanQ69L, precipitated with methanol-chloroform, and analyzed by SDS-PAGE and immunoblotting with indicated antibodies. Unspecific binding to Sepharose beads was analyzed in the absence of zz-transportin (-). For each immunoblot, the eluate and input was quantified by the ImageQuante software (GE Healthcare). The fraction of eluate compared with input (in %) is indicated at the bottom of each immunoblot. The band just below FUS is probably a degradation product of zz-transportin, which reacts with the antibody because of its tag.