Structure of the pre-mRNA leakage 39-kDa protein reveals a single domain of integrated zf-C3HC and Rsm1 modules

Abstract

In Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 Å resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP repeat)-like super family, with key residues of the canonical BIR domain being conserved. Features unique to the Pml39 modules refer to the spacing between the Zn-coordinating residues, giving rise to a substantially tilted helix αC in the zf-C3HC and Rsm1 modules, and an extra helix αAB' in the Rsm1 module. Conservation of key residues responsible for its distinct features identifies S. pombe Rsm1 and Homo sapiens NIPA/ZC3HC1 as structural orthologs of ScPml39. Based on the recent functional characterization of NIPA/ZC3HC1 as a scaffold protein that stabilizes the nuclear basket of the NPC, our data suggest an analogous function of ScPml39 in S. cerevisiae.

Figure 1

ScPml39 contains a single domain that recapitulates subcellular localization, function, and ScMlp1-interaction of full-length ScPml39. (a) Limited proteolysis of recombinant full-length ScPml39 at the indicated dilutions of 2 mg/ml elastase analyzed by SDS-PAGE. The full-length version of the cropped gel is presented in Fig. S4a. (b) Expression of GFP-tagged full-length (1–334) or truncated (77–317) versions of ScPml39 in pml39Δ cells detected by immunoblotting using anti-GFP antibodies. The full-length version of the cropped blot is presented in Fig. S4b. (c) Live imaging of pml39Δ cells expressing GFP-tagged full-length (1–334) or truncated (77–317) versions of ScPml39. Single plane images are shown for the GFP and DIC (differential interference contrast) channels. Arrowheads point to nuclei showing the U-shaped perinuclear staining typical of ScPml39. Scale bar, 5 µm. (d) Cells of the indicated genotypes were spotted as serial dilutions on SC medium and grown for 3 days at 25 °C. (e) Recombinant ScMlp1_1–325 forms a homodimer. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) using a Superdex 200 10/300 column was used. Molecular mass determination and Rayleigh ratio of ScMlp1_1–325 (dark and light gray, respectively) demonstrated that the ScMlp1_1–325 (expected molecular size is 41 kDa) has a dimeric size (~ 80 kDa). (f) Isothermal titration calorimetry (ITC) thermogram (upper panel) and plot of corrected heat values (lower panel) showed that monomeric ScPml39_77–317 binds dimeric ScMlp1_1–325 at a 1:1 molar ratio with a K_D value of ~ 13 μM.

Figure 2

Crystal structure of ScPml39_77–317. (a) Schematic of ScPml39. The consensus sequence of the zf-C3HC and Rsm1 modules is shown on the top. The zf-C3HC and Rsm1 modules are indicated in blue and purple, respectively. The fragment used for crystallization (residues 77–317) is shown. (b) Structure-guided sequence alignment of ScPml39, SpRsm1 and human NIPA/ZFC3HC1. αA–αE refer to α-helices, and β1–β3 to β-strands, indicating the secondary structure elements of ScPml39. Residue numbering is shown for ScPml39. Residues highlighted designate conserved Arg (magenta) and Ser/Thr (gray) in αA, Gly-aromatic residues between αB and β1 (green), conserved zinc-coordinating residues (yellow), a conserved aromatic residue in αD or in the loop αC'-αD' (cyan), and conserved hydrophobic residues in αAB' (brown). Similar and identical residues are marked as : and |, respectively. Disordered regions are represented by dotted lines, whereas regions lacking in the crystallization fragment are indicated by dashed lines and lowercase letters. (c) The Pml39 fold (cartoon representation) and side chains of key residues (stick representation) are shown.

Figure 3

Hydrophobic interface between zf-C3HC and Rsm1 modules. (a) Residues involved in van der Waals’ contacts are shown in stick representation. (b) Hydrophobicity of Rsm1 and zf-C3HC module are shown using PyMOL script color_h, ranging from white (highly hydrophobic) to green (less hydrophobic).

Figure 4

Structure of ScPml39 modules. (a) Superimposition of ScPml39 zf-C3HC module (in blue) and a representative canonical BIR domain, D. melanogaster IAP1-BIR1 domain structure (in orange, PDB: 3SIP) (left panel) and a view rotated by ~ 90° (right panel). (b) Structural conservation of Arg89 in helix αΑ and Trp178 in helix αD. (c) Internal hydrophobic network in ScPml39 zf-C3HC module. (d) Superimposition of ScPml39 Rsm1 module (in purple) and a representative canonical BIR domain, D. melanogaster IAP1-BIR1 domain structure (in orange, PDB: 3SIP) (left panel) and a view rotated by ~ 90° (right panel). (e) Structural conservation of Arg202 in helix αΑ′ and Tyr294 in helix αD′. (f) Internal hydrophobic network in ScPml39 Rsm1 module. (g) 2FoFc electron density, contoured at 1σ above the mean for helix αAB′ in Rsm1 module. (h) Internal hydrophobic network in ScPml39 Rsm1 module helix αAB′. (i) Different view from (h).