Molecular composition of staufen2-containing ribonucleoproteins in embryonic rat brain

Abstract

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (alpha- and beta-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.

Figure 1. Immunoprecipitation of Stau2 isoforms.

(A) Schematic representation of Stau2 isoforms. The Stau2 gene generates four different isoforms of 62, 59, 56 and 52 kDa through differential splicing. Black, grey and white boxes represent double-stranded RNA-binding (dsRBD) consensus sequence having full, partial or no RNA-binding activity, respectively. Hatched boxes represent the tubulin-binding domain (TBD). (B) Immunoprecipitation of Stau2 isoforms from embryonic E17-18 rat brain extracts using two different polyclonal anti-Stau2 antibodies, L1 (St2-L1) and L2 (St2-L2). The specificity of these antibodies was previously reported . Pre-immune (PI) sera were used as controls. The Stau2⁵⁶ isoform is not visible in these cell extracts. * represents a non-specific IgG band.

Figure 2. RNA-binding proteins are associated with Stau2 isoforms in mRNPs.

N2A cells were mock transfected (−) or co-transfected with plasmids coding for either Stau2⁵⁹-HA₃ (59) or Stau2^62- HA₃ (62) and plasmids coding for PABPC1-myc (A), YB1-CFP (B), hsc70-CFP (C) or hnRNP H1-myc (D) as indicated. Immunoprecipitation of Stau2-containing RNPs was performed with anti-HA antibody and the proteins detected on western blots using anti-HA, anti-myc or anti-GFP antibodies as needed. The experiments were done in the absence (-RNase) or presence (+RNase) of Microccocal nuclease to determine if the Stau2-protein association requires an RNA bridge. These results were representative of at least three experiments. Input (INPUT) of transfected proteins before immunoprecipitation is also shown to indicate that the tagged-proteins were well expressed in these cells.

Figure 3. MBP-Stau2⁶² binds GST-hsc70 through protein-protein interaction.

To confirm the RNA-resistant interaction between Stau2 and hsc70, bacterially expressed proteins were purified (A) and GST-pull down (B) and surface plasmon resonance SPR (C) assays were performed in the presence or absence of RNase A. (A) MBP-Stau2⁶², MBP, GST-hsc70 and GST were purified on amylose and glutathione-Sepharose-4B affinity columns, respectively, and eluted proteins were analyzed by SDS-PAGE and Coomassie brilliant blue staining. (B) GST and GST-hsc70 were fixed on a glutathione-Sepharose-4B affinity column and MBP-Stau2⁶² was loaded in the presence (+) or absence (−) of RNase A. After several washing, proteins were eluted from the columns and detected by western blotting using anti-Stau2 and anti-GST antibodies, respectively. (C) MBP and MBP-Stau2⁶² were immobilized on different lanes of a SPR sensor chip. GST-hsc70 or GST were injected for 3 minutes over the surfaces in the presence or absence of RNase and then buffer alone was injected for 2.5 min to monitor protein dissociation rate. The resulting resonance units (RU) were measured during the association and dissociation phases. The baseline obtained with the MBP-coupled reference surface was subtracted from the sensorgram obtained from the MBP-Stau2⁶²-coupled surface and a typical result is shown.

Figure 4. The interaction between Stau2⁶² and hsc70 is abolished in the presence of ATP.

(A) N2A cells were co-transfected with plasmids coding for Stau2^62- HA₃ and hsc70-CFP as done for figure 2. Immunoprecipitation of Stau2-containing RNPs was performed with anti-HA antibody and the proteins detected on western blots using anti-HA or anti-GFP antibodies as needed. The experiment was done in the absence (−) or presence of either Microccocal nuclease (+RNase) or ATP (+ATP). These results are representative of at least three experiments. (B) As done for figure 3C, MBP and MBP-Stau2⁶² were immobilized on different lanes of a SPR sensor chip. GST-hsc70 or GST was injected for 3 minutes over the surfaces in the presence or absence of ATP. The baseline obtained with the MBP-coupled reference surface was subtracted from the sensorgram obtained from the MBP-Stau2⁶²-coupled surface and a typical result is shown.

Figure 5. Co-localization of endogenous Stau2 with PABPC1-myc, YB1-CFP, hsc70-CFP and hnRNP H1-myc in hippocampal neurons.

Neurons were transfected with plasmids coding for either PABPC1-myc (A), YB1-CFP (B), hsc70-CFP (C) or hnRNP H1-myc (D). Twenty four hours post-transfection, neurons were fixed and labeled with anti-myc or anti-GFP (green) and anti-Stau2 (red) antibodies. Left: Fluorescence microscopy of hippocampal neurons in culture. Scale bars: 5 µm. Right: Higher magnification of images showing protein localization in dendrites. The lower panels represent the superposition of both green and red signals. Scale bars: 2 µm.

Figure 6. Co-localization of Stau2⁶²-HA₃ with endogenous PABPC1 and YB1 in hippocampal neurons.

Neurons were transfected with a plasmid coding for Stau2⁶²-HA₃. Twenty four hours post-transfection, neurons were fixed and labeled with anti-HA antibody (red) and either anti-YB1 (A) or anti-PABPC1 (B) antibodies (green). Left: Fluorescence microscopy of hippocampal neurons in culture. Scale bars: 5 µm. Right: Higher magnification of images showing protein localization in dendrites. The lower panels represent the superposition of both green and red signals. Scale bars: 2 µm.