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. 2012 Oct;40(18):9356-68.
doi: 10.1093/nar/gks696. Epub 2012 Jul 24.

NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold

Urszula M Wolkowicz  1 , Atlanta G Cook
Affiliations

NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold

Urszula M Wolkowicz et al. Nucleic Acids Res. 2012 Oct.

Abstract

Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes and are thought to affect gene expression both at the transcriptional and translational level. In addition, this complex affects the replication of several viruses through direct interactions with viral RNA. NF90 and NF45 dimerize through their common 'DZF' domain (domain associated with zinc fingers). NF90 has additional double-stranded RNA-binding domains that likely mediate its association with target RNAs. We present the crystal structure of the NF90/NF45 dimerization complex at 1.9-Å resolution. The DZF domain shows structural similarity to the template-free nucleotidyltransferase family of RNA modifying enzymes. However, both NF90 and NF45 have lost critical catalytic residues during evolution and are therefore not functional enzymes. Residues on NF90 that make up its interface with NF45 are conserved in two related proteins, spermatid perinuclear RNA-binding protein (SPNR) and zinc-finger RNA-binding protein (Zfr). Using a co-immunoprecipitation assay and site-specific mutants, we demonstrate that NF45 is also able to recognize SPNR and Zfr through the same binding interface, revealing that NF45 is able to form a variety of cellular complexes with other DZF-domain proteins.

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Figures

Figure 1.
Figure 1.
Overview of the NF90/NF45 DZF dimerization domain structure. (A) Schematic representation of the domain organization of NF45 (green) and NF90 (blue). Double-headed arrows underneath indicate the sub-fragments of each protein that were used for crystallization. (B) Overview of the structure showing a ‘side’ view and a ‘top’ view rotated 90° around the horizontal axis. (C) Superposition of NF90 (blue) on NF45 (gray) using the same orientations of NF45 as shown in B. Extensions at the N- and C-termini of NF45 that are involved in dimerization interactions are shown in yellow. A C-terminal extension of NF90 is shown in magenta with a dotted line indicating a missing loop. (D) Superposition of NF45 (grey) on yeast Poly (A) polymerase (PDBid 2q66, brown) and CCA-adding enzyme from Archaeoglobus fulgidis (PDBid 1tfw, pink), shown from the ‘top’ view from (B).
Figure 2.
Figure 2.
Conservation of NF90 and NF45 sequences. (A) Multiple sequence alignment of NF45 with sequences from mouse (M.m) zebrafish (D.r.) Drosphila (D.m.) and Ciona intestinalis (C.i.). A structure-based alignment of NF45 with yeast poly (A) polymerase is shown underneath. Human NF45 was not included as it has 100% identity with mouse NF45. Secondary structure elements are shown underneath as bars (α-helices) or arrows (β-strands). Blue dots indicate residues that interact with NF90, pink dots indicate residues that interact with ATP and black boxes show residues equivalent to those involved in catalysis in functional nucleotidyl transferases. Purple boxes indicate the two residues on helix 9 that were mutated to disrupt dimer formation. (B) Sequence alignment of NF90 with sequences from mouse (M.m.), human (H.s.), zebrafish (D.r.) and Xenopus (X.l.). NF90 is only found in vertebrates. Green dots indicate residues that are involved in dimerization interaction with NF45.
Figure 3.
Figure 3.
A close-up view of the active site of poly (A) polymerase and equivalent regions in NF90 and NF45. (A) The catalytic cleft of yeast poly (A) polymerase (derived from PDBids 2q66 and 3c66) showing the catalytic residues and the bound ATP and a poly (A) substrate. (B) and (C) top view of NF45 and NF90 oriented as in Figure 1C, looking into the cleft between the two domains. The residues shown are those in equivalent positions to catalytic and nucleotide-binding residues found in related nucleotidyl transferases. (D) ATP bound to the cleft of NF45. A 2Fo–Fc map from the final refined structure is shown, contoured at 1σ. (E) and (F) A similar view as in (D) showing complexes with UTP and CTP, respectively.
Figure 4.
Figure 4.
The NF90/NF45 dimerization interface. (A) Close up view of the dimerization interface in the same orientation as the top view in Figure 1B. The C-terminal extension of NF45 (yellow) lies in an extended conformation over helix 9 (H9) of NF90 (blue) and NF45 (green). Helix 9 shows symmetrical binding interactions at the core. Mutated residues in NF45 are marked with an asterisk. (B) A close up view of the interface rotated 180° around the horizontal axis. The N-terminal extension of NF45 is shown in yellow. Conserved residues involved in the dimerization interface and in stabilizing the N-terminal extension are shown. (C) A sequence alignment of the dimerization interface of mouse NF90 with related DZF domain-containing proteins, SPNR and Zfr. Sequences from mouse (M.m.), zebrafish (D.r.) Xenopus species (X.l. and X.t.) and salmon (S.s.) are shown.
Figure 5.
Figure 5.
Co-immunoprecipitation studies of NF45 with other DZF domain-containing proteins. (A) Overview of domain organization of DZF-domain containing proteins. (B) Co-immunoprecipitation studies carried out with HA-tagged proteins (1. HA-NF45, 2. HA-NF90, 3. HA-STRBP) and with differentially tagged pairs of NF45 (lane 4) and NF90 (lane 5). (C) Co-immunoprecipitation studies of NF45 wild-type (lanes 1, 3, 4 and 6) or NF45 mutant (lanes 2, 5 and 7) co-transfected with NF90 (lanes 1 and 2), SPNR (lanes 3,4 and 5) and Zfr (lanes 6 and 7).
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