Fibroblast growth factor-2(23) binds directly to the survival of motoneuron protein and is associated with small nuclear RNAs - PubMed (original) (raw)

Fibroblast growth factor-2(23) binds directly to the survival of motoneuron protein and is associated with small nuclear RNAs

Peter Claus et al. Biochem J. 2004.

Abstract

The SMN (survival of motoneuron) protein is mutated in patients with the neurodegenerative disease spinal muscular atrophy. We have shown previously that a high-molecular-mass isoform of FGF (fibroblast growth factor) 2 (FGF-2(23)) is in a complex with SMN [Claus, Doring, Gringel, Muller-Ostermeyer, Fuhlrott, Kraft and Grothe (2003) J. Biol. Chem. 278, 479-485]. FGF-2 is a neurotrophic factor for motoneurons, and is known not only as a classical extracellular growth factor, but also as a nuclear protein. In the present study, we demonstrate that SMN binds to the arginine-rich N-terminus of FGF-2(23). In turn, FGF-2(23) interacts with amino acid residues 1-90 of the human SMN protein. This sequence displays nucleic-acid-binding capacity and overlaps partially with known binding sites for Gemin2/SIP1 (SMN-interacting protein 1) and p53. Finally, as a functional consequence of FGF-2(23) binding to SMN, FGF-2(23) is in a complex with the small nuclear RNAs U2 and U4. Since SMN functions as an assembly factor for snRNPs (small nuclear ribonucleoprotein particles), these results suggest binding of FGF-2(23) to snRNPs.

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Figures

Figure 1. SMN binds to the arginine-rich N-terminus of FGF-223 in vitro, but not to other FGF-2 isoforms

(a) Recombinantly expressed FGF-2 isoforms (18, 21 and 23 kDa) were immobilized on a nitrocellulose membrane and probed with in vitro translated SMN (far-Western blot). SMN binding was subsequently analysed using anti-SMN antibody and chemiluminescence detection. SMN bound to the FGF-223 isoform, but not to the smaller isoforms, demonstrating that the number or configuration of RGR-motifs is critical for efficient binding. (b) The sequence shows the N-termini of FGF-223 and FGF-221 with RGR motifs and hydrophobic spacer sequences. Numbers denote length of spacers as numbers of amino acid residues. Thick lines with arrows, length of the N-termini 21 and 23 kDa isoforms of FGF-2 respectively with translation starts at CUG codons (leucine). FGF-218 starts with a methionine residue from an AUG codon.

Figure 2. The SMN–FGF-223 complex is RNase-resistant

(a) Co-immunoprecipitations with an anti-FGF-2 polyclonal antibody of lysates from Schwann cells overexpressing FGF-223 revealed no change of SMN binding to FGF-223 upon extensive digestion with RNase A. Immunoprecipitation (IP) with anti-SMN antibody is a control. Detection antibody for the Western blot was anti-SMN. (b) Control for the specificity of the employed anti-FGF-2 polyclonal antibody. Lysates from FGF-218-overexpressing Schwann cells were subjected to immunoprecipitations with anti-FGF-2. Detection with anti-SMN revealed only a very weak band due to the presence of endogenous FGF-223. HC, heavy chain; LC, light chain of IP antibodies.

Figure 3. FGF-223 binds directly to a N-terminal sequence of SMN

(a) For in vitro binding experiments (pull-downs) SMN-deletion mutants were expressed as fusion proteins with GST, bound to glutathione–agarose beads and incubated with recombinant FGF-223. After the binding reaction, beads were washed with IP buffer and PBS, and the bound FGF-223 was detected by SDS/PAGE and Western blot with anti-FGF-2 antibody. To demonstrate equal loading, GST–SMN mutants were stained with Coomassie Blue. (b) Pull-down experiment similar to that of (a), but, instead of recombinant FGF-2, lysates from Schwann cells overexpressing DsRed-tagged FGF-223 were used. After 1 h of binding, beads were washed extensively with IP buffer (enriched with 1 M NaCl, 0.1% Tween 20 and 0.1% Nonidet P40) and the bound protein was analysed by SDS/PAGE and Western blot with anti-FGF-2 antibody. As a control, the same amount of beads employed as in the binding reactions (visual estimation after Coomassie Blue staining) were Coomassie-Blue-stained after SDS/PAGE. The aligned bands are shown as loading controls. Numbers denote the amino acid residues of the SMN mutants. (c) Structure of SMN-deletion mutants (boxes) and results of pull-downs. C-terminal end of binding site: FGF-223 bound to SMN 1–90 as the minimal fragment of the N-terminus. The 28–294 SMN fragment was only weakly bound, indicating the importance of the sequence from residues 1–28 as a part of the binding site. The compared data demonstrate FGF-223 binding to a N-terminal sequence of SMN, which is coded by exons 1–3 of the human SMN gene. The sequence codes by exons 2 and 3 demonstrates homology with the so-called HMGB-box of HMGB proteins and HMGB-box-containing proteins, and was shown previously to interact in vitro with nucleic acid homopolymers [38]. The relative binding intensity is indicated as +, (+) and −. The positions of the Tudor domain and the C-terminal domain, which is deleted in most cases of SMA, are shown.

Figure 4. U2 snRNA binding to SMN is not influenced by FGF-223

Immobilized full-length SMN as a fusion with GST was incubated with labelled U2 snRNA, unlabelled non-specific competitor [tRNA (results not shown) or poly (dG-dC)·poly(dG-dC)] and no or increasing amounts of recombinant FGF-223 (lanes 1–5). After incubation, beads were washed, the RNA was recovered by TRIzol® isolation and detected after dot blotting of bound fractions. Only a very high amount of FGF-223 was able to disrupt RNA–SMN binding. No non-specific U2 snRNA binding to beads could be detected (lanes 6 and 8). Immobilized FGF-223 did not bind to U2 snRNA.

Figure 5. FGF-223 is associated with RNA from snRNPs

FGF-223 complexes were immunoprecipitated from lysates of Schwann cells. RNA was purified by a combination of two methods, reverse-transcribed with specific primers for snRNAs (U2, U4 and U6 snRNAs respectively) and subsequently analysed by PCR. As a control, RNA from input material was used and reverse-transcribed to show the presence of all U snRNAs in the input, as well as to have a loading-control. Additionally, primers for the housekeeping gene GAPDH were employed as combined controls for DNA contamination and non-specifically bound RNA molecules respectively. U2 and U4, but not U6, snRNAs are associated with the FGF-223 complex, indicating a role for this FGF-2 isoform in the association with snRNPs from the cytosol. Unlike U2 and U4 snRNAs, U6 snRNAs do not leave the nucleus for snRNP assembly. Controls with GAPDH does not show non-specifically bound mRNA (IP, immunoprecipitation) or DNA contamination (input).

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Fibroblast growth factor-2(23) binds directly to the survival of motoneuron protein and is associated with small nuclear RNAs - PubMed (original) (raw)