The microtubule-associated protein tumor overexpressed gene binds to the RNA trafficking protein heterogeneous nuclear ribonucleoprotein A2

Abstract

In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization.

Figure 1.

Identification of TOG isoforms. (A) The top black line represents the sequence (residues 829-1564... 1623–1957) used to raise antibodies in rabbits (Charrasse et al., 1998) (Rb-anti-TOG), whereas the displayed sequence to the left indicates the oligopeptide used to raise antibodies in chickens (Ck-anti-TOG) and its location (529–545). The top black bar, ch-TOG, is a map of the small isoform of TOG encoded by the TOG gene (GenBank accession no. D43948). The white boxes indicate the locations of HEAT repeats (Neuwald and Hirano, 2000; Andrade et al., 2001); the diagonally striped box represents the location of an exon encoding 60 residues (1564–1623) present in the large isoform of TOG, TOG2 (lowest black bar) but absent in ch-TOG. The middle black bar represents the C-terminal portion of TOG2, TOG2-C, that binds to hnRNP A2. (B) Positions and directions of primers (A–D, identified by arrows) designed to distinguish ch-TOG from TOG2 mRNA (see Results) based on the presence or absence of the exon (residues 1564–1623). (C) RT-PCR was performed using sets of primers (described above and in Table 1 in Supplementary Material) to identify both ch-TOG and TOG2 (AC) and to distinguish ch-TOG (CD) and TOG2 (AB) mRNAs in cells. mRNA from B104, oligodendrocytes (OLGs), and U-2 OS cells was amplified with LKTOG_A and LKTOG_C (AC), LKTOG_C and LKTOG_D (CD), or LKTOG_A and LKTOG_B (AB). The size of the bands is estimated from the pattern of a 100-base pair DNA ladder (right).

Figure 2.

HnRNP A2/TOG interactions. (A) Autoradiogram of a PAGE of in vitro transcription-translation reactions: [³⁵S]methionine-labeled proteins encoded by the bait (pBD+hnRNPA2), hnRNP A2, and two pAD-cDNA positive clones, HA-hnRNP A2 and HA-TOG2-C. Molecular mass standards in kilodaltons are shown on the left. (B) Autoradiogram of the immune precipitates of the proteins described in A by using anti-HA antibodies (see Materials and Methods). (C) Coimmunoprecipitation of the [³⁵S]methionine-labeled hnRNP A2 described in A and cold HA-hnRNP A2 and HA-TOG2-C by using anti-HA antibodies. The reaction was carried out at various salt concentrations as indicated. (D) An equal amount of a B104 cellular extract was reacted with an anti-TOG IgY fraction or with a preimmune IgY fraction of the same concentration. Immune complexes were precipitated and analyzed as described in Materials and Methods. The same volume of the immune and preimmune reaction mixes was applied to the gel. The presence of hnRNP A2 in the immune complexes was revealed by Western blot by using a monoclonal anti-hnRNP A2 murine antibody (Nichols et al., 2000). The exposure time necessary to detect hnRNP A2 in the preimmune pellet results in a band of saturated intensity in the anti-TOG pellet.

Figure 3.

TOG association with the cytoskeleton. (A) Primary cultures of oligodendrocytes (OLGs) and neurons were treated with detergent-containing buffer (see Materials and Methods) to remove cellular components that are not associated with the CSK. This fraction (sol) and the remaining nonextracted components (ins) were run on a gel and probed for the presence of TOG by Western blotting by using chicken anti-TOG antibodies. The size of the cross-reacting peptides was calculated by comparing their migration pattern to that of standard proteins and is indicated to the left. (B) CSK-extracted oligodendrocytes were fixed and reacted with anti-TOG (left) and anti-α-tubulin (right). Bar, 12 μm. High-magnification view of a distal process of the oligodendrocyte in B: anti-TOG (red), anti-α-tubulin (green). (C) Western blot of immune complexes from a CSK extract of oligodendrocytes reacted with anti-TOG or preimmune serum, and probed with anti-hnRNP A2. (D) Western blot of a microtubule-stabilized pellet from B104 cells probed with anti-TOG, anti-hnRNP A2, and anti-β-tubulin.

Figure 4.

TOG association with subcellular components. Representative images of oligodendrocytes that were fixed, permeabilized, and immunostained for TOG (A, D, G, and J), CLIP 115 (B), hnRNP A2 (E), and hnRNP K (H) or injected with fluorescent UTP-labeled A2RE RNA (K). Green channel: A, D, G, and J and pairwise red channel: B, E, H, and K. TOG/CLIP (A–C), TOG/hnRNP A2 (D–F), TOG/hnRNP K (G–I), and TOG/A2RE RNA (J–L). Insets in B, E, H, and K: enlargements of a portion of cellular processes visualized as merged channels (A and B, D and E, G and H, and J and K, respectively). Yellow indicates spatial coincidence in the green and red channels. Bars, 20 μm (A and B), 15 μm (D and E), 12 μm (G and H), and 10 μm (J and K). Single granule ratiometric analysis (C, F, I, and L) was performed on cells treated as described above by integrating the intensities in the green channel (A, D, G, and J) and red channel (B, E, H, and K), respectively, for each granule. Only well resolved granules located in the medial and distal portions of the cellular processes were analyzed. Each dot represents the coordinates of one granule. Number (n) of granules analyzed: n = 3647 (C), n = 3217 (F), n = 1854 (I), and n = 1897 (L).