The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export - PubMed (original) (raw)

The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export

Deanna M Green et al. Proc Natl Acad Sci U S A. 2003.

Abstract

For mRNA to be transported from the nucleus to the cytoplasm, it must travel from the site of transcription through the nuclear interior to the nuclear pore. Studies in Saccharomyces cerevisiae have suggested a relationship between poly(A) RNA trafficking and myosin-like protein 1 (Mlp1p), a nuclear-pore associated protein that is homologous to the mammalian Tpr (translocated promoter region) protein [Kosova, B., Panté, N., Rollenhagen, C., Podtelejnikov, A., Mann, M., Aebi, U., and Hurt, E. (2000) J. Biol. Chem. 275, 343-350]. We identified a yeast two-hybrid interaction between the C-terminal globular domain of Mlp1p and Nab2p, a shuttling heterogeneous nuclear ribonucleoprotein that is required for mRNA export. Coimmunoprecipitation confirms that Nab2p also interacts with full-length Mlp1p and in vitro binding experiments show that Nab2p binds directly to the C-terminal domain of Mlp1p. In addition, our experiments reveal that the C-terminal domain of Mlp1p is both necessary and sufficient to cause accumulation of poly(A) RNA and Nab2p in the nucleus. We propose a model where Mlp1p acts as a checkpoint at the nuclear pore by interacting with export-competent ribonucleoprotein complexes through its C-terminal globular domain. This study identifies Nab2p as a heterogeneous nuclear ribonucleoprotein found in complex with Mlp1p and begins to delineate the path that mRNA travels from the chromatin to the nuclear pore.

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Figures

Figure 1

Figure 1

Nab2p directly interacts with CT-Mlp1p. (A) Cells expressing DBD-Nab2p and pJG4–5 vector, AD-Mlp1p and pEG202 vector, or DBD-Nab2p and AD-Mlp1p (Both) were grown on galactose synthetic complete (SC) or galactose plates lacking leucine (−leu). (B) Schematic of the domains and NLS (23) of Mlp1p and the fragment identified in the yeast two-hybrid screen (AD-Mlp1). Amino acid residues are designated below each schematic. (C) Immunoblot analysis of protein lysates containing either Nab2p-myc (lanes 1 and 2) or control vector (lane 5) and either GFP control (lane 1) or CT-Mlp1p-GFP (lanes 2 and 5). Lysates were immunoprecipitated with anti-myc Ab and bound fractions were probed with anti-GFP Ab (Top and Middle) or anti-myc Ab (Bottom) (lanes 3, 4, and 6). (D) Immunoblot analysis of protein lysates from WT (lane 1) or Mlp1p-protein A (lane 3) strains. Proteins were immunoprecipitated with IgG-Sepharose and bound fractions were probed simultaneously with anti-rabbit Ab and anti-Nab2p (lanes 2 and 4). (E) GST (lanes 5 and 6) and GST-CT-Mlp1p (lanes 1–4) were incubated with recombinant Srp1p (lanes 3 and 4) or Nab2p (lanes 1, 2, 5, and 6) and unbound (U) and bound (B) fractions were analyzed by immunoblotting.

Figure 2

Figure 2

Characterization of the CT-Mlp1p. (A) Cells expressing a galactose-inducible vector (A_–_C), FL-Mlp1p (D_–_F), CT-Mlp1p (G_–_I), or ΔCT-Mlp1p (J_–_L) were grown in galactose and poly(A) RNA was visualized by fluorescence in situ hybridization analysis. Corresponding DAPI staining and merged images are shown. DAPI, green; poly(A) RNA, red; merge, yellow. (B) Cells expressing a galactose-inducible CT-Mlp1p-GFP protein were grown in galactose and CT-Mlp1p-GFP and poly(A) RNA were visualized by indirect immunofluorescence (A) and fluorescence in situ hybridization analysis (B). Corresponding merged (C) and differential interference contrast (DIC) (D) images are shown. GFP, green; poly(A) RNA, red; merge, yellow. (Magnification: ×100.)

Figure 3

Figure 3

Overexpression of Mlp1p inhibits cell growth. WT cells containing a galactose-inducible vector control, FL-MLP1 (FL), CT-MLP1 (CT), or Δ_CT-MLP1_ (ΔCT) were grown to saturation in glucose, serially diluted, and spotted onto glucose (Left) or galactose (Right) minimal media plates.

Figure 4

Figure 4

Overexpression of CT-Mlp1p causes Nab2p accumulation within the nucleus. (A) GFP signal was visualized in WT cells (A and C) and rat7-1 cells shifted to 37°C (B and D) expressing Nab2p-GFP (A and B) or ΔRGG-Nab2p-GFP (C and D). Corresponding differential interference contrast images are shown. (B) GFP signal was visualized in WT cells that express a vector control (A and E), FL-Mlp1p (B and F), CT-Mlp1p (C and G), or ΔCT-Mlp1p (D and H) and coexpress ΔRGG-Nab2p-GFP (A_–_D) or Nab2p-GFP (E_–_H). Corresponding differential interference contrast images are shown. (Magnification: ×100.)

Figure 5

Figure 5

CT-Mlp1p coimmunoprecipitates Npl3p. Immunoblot analysis of protein lysates containing Npl3p-myc (lanes 1 and 2) or Srp1p-myc (lane 5) and GFP control (lane 1) or CT-Mlp1p-GFP (lanes 2 and 5). Proteins were immunoprecipitated with anti-myc Ab and bound fractions were probed with anti-GFP Ab (Top and Middle) or anti-myc Ab (Bottom) (lanes 3, 4, and 6).

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