Complex formation of the neuron-specific ELAV-like Hu RNA-binding proteins

Abstract

Hu proteins are RNA-binding proteins that are the vertebrate homologs of Drosophila ELAV, and are implicated in stabilization or enhanced translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Here, using the yeast two-hybrid system, we show that neuron-specific Hu proteins can interact with themselves. Immuno precipitation assays demonstrated that the interaction between Hu proteins occurs in mammalian cells and is strongly enhanced in the presence of cellular RNA. Furthermore, using in situ chemical crosslinking assays, we found that HuD, one of the neuron-specific Hu proteins, multimerizes in cells. The crosslinked HuD multimers retain specific RNA-binding ability and can interact with additional Hu proteins. Consistent with this biochemical property, HuD showed granular distribution in two neurogenic cell lines. These results suggest that the RNA-bound form of HuD multimerizes cooperatively to form a specific granular structure that may serve as a site of post-transcriptional regulation of ARE-containing mRNAs.

Figure 1

Protein–protein interaction between Hu proteins. (A) The results of yeast two-hybrid analysis using neuron-specific Hu proteins. The β-galactosidase activity of transformants expressing each combination of proteins is normalized by the activity of the combination of vector plasmids (pAS2-1 and pACT2): –, <3-fold; +, 3–10-fold; ++, 10–20-fold; +++, >20-fold over the control value. HuC* and pTD1-1 represent human HuC lacking the N-terminal 20 amino acid residues and the negative control plasmid encoding SV40 large T antigen, respectively. (B) In vitro binding of ³⁵S-methionine-labeled HuD to the indicated recombinant GST–Hu fusion proteins in the presence of RNase A. (C) Hu proteins can self-associate in mammalian cells. HeLa cells were co-transfected with FLAG–HuD and either T7–HuD, T7–HuB or T7–HuC. The cell extracts were immunoprecipitated with anti-FLAG antibody (FLAG–IP) or anti-T7 antibody (T7–IP), and the precipitants were analyzed by western blotting with anti-T7 antibody (α-T7) or anti-FLAG antibody (α-FLAG). The asterisks indicate the heavy and light chains of the IgG used for immunoprecipitation. As controls, the levels of each proteins in the cell extracts used are shown below (input: α-FLAG, α-T7).

Figure 2

RBDs are responsible for self-association of Hu proteins. (A) Schematic representation of the fragments of HuC used for the yeast two-hybrid analysis. RBDs are represented by boxes. (B) The relative β-galactosidase activity of the transformants expressing HuB fused to the DNA-binding domain (BD) and various fragments of HuC fused to the activation domain (AD). The β-galactosidase activity was normalized by the activity of the combination of HuB and HuC. (C) The relative β-galactosidase activity of the transformants expressing CRBD3 fused to BD and various fragments of HuC fused to AD. The β-galactosidase activity was normalized by the activity of the combination of CRBD3 and HuC.

Figure 3

Cellular RNA is required for efficient interaction between HuD molecules. (A) Scheme of the in vitro binding experiment using HeLa cell extracts containing T7–HuD and FLAG–HuD. (B) Immunoprecipitation of the mixed extracts with anti-T7 antibody in the presence or absence of RNase A. RNase treatment had no effect on the levels of tagged HuD in the extract (two bottom panels). The asterisks indicate the heavy and light chains of IgG used for immunoprecipitation. (C) Binding of wild-type HuD and a mutant HuDmt to the poly(U) sequence. HeLa cell extracts containing T7–HuD or T7–HuDmt were incubated with poly(U)–Sepharose beads and the bound proteins were analyzed by western blotting with anti-T7 antibody. As controls, the levels of T7–HuD and T7–HuDmt in the extracts used are shown (input). (D) Pull-down of the in vitro translated myc-HuD or myc-HuDmt by GST or GST–HuD. Proteins pulled-down by GST or GST–HuD were analyzed by western blotting with anti-myc antibody. As controls, the levels of myc-HuD and myc-HuDmt in the in vitro translation reactions used are shown (input).

Figure 4

Complex formation of neuron-specific Hu proteins in mammalian cells revealed by in situ chemical crosslinking. PC12 cells expressing each T7–Hu protein were treated with BMH or DSS, and the cell extracts were then analyzed by western blotting using anti-T7 antibody. (A) Formation of dimer and trimer complexes of HuD. T, D and M indicate trimer, dimer and monomer of HuD, respectively. X indicates a specific HuD complex that has yet to be identified. (B) Crosslinked complexes of deletion constructs of HuD, RBD1-del and RBD3-del. T′, D′ and M′ indicate the trimer, dimer and monomer of them, respectively. X′ corresponds to the X complex of HuD. (C) The results of in situ crosslinking with T7–HuB and T7–HuC. (D) The results of in situ crosslinking with T7–ELAV or FLAG–HuR. (E) Cysteine residues within Hu proteins and ELAV. The asterisks indicate the cysteine residues that are the possible sites for chemical crosslinking by BMH.

Figure 5

Multimeric HuD complexes retain RNA-binding activity and interact with Hu proteins. (A) Binding of multimeric HuD complexes to poly(U) RNA. HeLa cells expressing T7–HuD were treated with BMH, and the cell extracts were then pulled down with poly(U)–Sepharose [poly(U)] or control Sepharose 4B (4B) beads. The bound proteins were analyzed by western blotting using anti-T7 antibody. (B) Co-precipitation of the crosslinked HuD complexes with HuD and HuB. HeLa cell extracts containing the crosslinked T7–HuD complexes were mixed with the cell extract containing FLAG–HuD or FLAG–HuB, and the mixed samples were then immunoprecipitated with anti-FLAG antibody, followed by western blotting with anti-T7 antibody (α-T7). As controls, the levels of FLAG–HuD and FLAG–HuB in the cell extracts used are shown below (α-FLAG).

Figure 6

Granular distribution of HuD in two neurogenic cell lines. Immunostaining views of PC12 (top) or SH-SY5Y (bottom) cells expressing T7–HuD (left) or T7–HuDmt (right). After transfection, cells were fixed and immunostained with anti-T7 antibody.