MT/Impα binding. Purified GST and GST–MT (A) or GST–Impα (B) were incubated with [³⁵S]Met-labeled Impα and MT, respectively. Bound proteins were detected by SDS-PAGE followed by autoradiography (Yue et al. 1997; Wen et al. 1998). HeLa S3 cells (C) were transfected with pEGFP-C1–Impα and pcDNA3.1(+)-MT-myc. Two days later, cell lysates were immunoprecipitated (IP) in RIPA buffer (see Materials and Methods) as indicated, with anti-myc (α-myc) or anti-GFP (α-GFP) antibodies, and immunoblotted with α-GFP or α-myc. (G) GFP only; (G-I) GFP–Impα fusion protein; (m) myc tag only; (M-m) MT–myc fusion protein.

Regions required for Impα/MT complex formation, MT activity, and RNA binding in vitro. Purified MT (A) or Impα (B) and the indicated truncation mutants fused with GST were incubated with [³⁵S]Met-labeled full-length Impα or MT, and bound proteins were detected by SDS-PAGE and autoradiography. MT activity was tested by incubating the indicated proteins with Adomet and T7 polymerase 32-nt runoff transcripts containing [³²P]GpppG-5′ ends, followed by P1 nuclease digestion, TLC, and autoradiography (Yue et al. 1997; Pillutla et al. 1998). RNA binding was measured using GpppG-primed T7 polymerase runoff transcripts labeled with [α-³²P]GTP (Furuichi and Shatkin 1994), and nucleoprotein complexes were separated by 4.5% PAGE at 4°C (Buratowski and Chodosh 1996) followed by autoradiography. The values corresponding to + and − were >80% and <10%, respectively, of the levels obtained with the full-length protein. (ND) Not done.

Effect of Ran on protein/RNA complex formation and cap methylation. (A) Samples containing 1 μM MT (lane 1) plus 1 μM Impα (lane 2), plus 3 μM Impβ (lane 3), plus 5μM RanGTP (lane 4) or RanGDP (lane 5) were incubated with GpppG-RNA and assayed by gel shift as in Fig. 3. (B) Samples containing 3 nM MT (column 1); 3nM MT, 30 nM Impα (column 2); 3 nM MT, 30 nM Impα, 120 nM Impβ (column 3); 3nM MT, 30 nM Impα, 120 nM Impβ, 200 nM RanGTP (column 4) or RanGDP (column 5), were assayed for cap methylation as in Fig. 4.

Localization of MT and Impα fusion proteins. HeLa S3 cells were transfected with pEGFP-C1–MT and pDsRed1-N1–Impα (A–C) or pEGFP-C1–MT (144–476) (D), constructed as recommended by Clontech, and analyzed by fluorescence microscopy.

Impα enhances MT selective binding to GpppG-RNA and cap methylation, which are both prevented by Impβ. (A) Purified MT was incubated with T7 polymerase 32-nt runoff transcripts labeled with [α-³²P]GTP and containing 5′-terminal GpppG, pppG, or m⁷GpppG (Furuichi and Shatkin 1994) (lanes 1–3, respectively) or 5′-teminal GpppG in the presence of 1mM cap analog, as indicated (lanes 4–6). The MT/RNA complexes were detected as in Fig. 3. (B) MT at 0 (lanes 1,6), 0.10 (lanes 2,7), 0.25 (lanes 3,8), 0.5 (lanes 4,9), or 1.0 (lanes 5,10) μM was incubated with GpppG-ended radiolabeled T7 polymerase runoff transcripts in the absence (−) or presence (+) of 1.0 μM Impα. (Lanes 11–13) Samples contained the GpppG-RNA, 1.0 μM (each) Impα and MT, and 1 mM cap analog as indicated. (Lanes 14–18) Impβ was added at concentrations of 0, 1, 2, 4, or 8 μM with purified MT and Impα (1.0 μM each) and GpppG-RNA. (Lanes 19,20) GpppG-RNA was incubated with 1.0 μM Impα (1–455) and either 0 or 1.0 μM purified MT, respectively. Nucleoprotein complexes were separated by PAGE, detected by autoradiography, and quantitated by PhosphorImager. (C) 3 nM MT was incubated with 0, 3, 6, 15, 30, 60, or 150 nM Impα (closed triangles) or Impα (1–455) (closed circles), and MT activity assays were performed as in Fig. 3. (D) 0, 30, 60, 120, or 240 nM Impβ was incubated with 3 nM MT and 30 nM Impα and assayed as above.