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. INTRODUCTIONThe heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is implicated in RNA processing, RNA transport, and translation regulation (Dreyfuss et al., 1993;Siomi and Dreyfuss, 1995;Hamilton et al., 1999;Kwon et al., 1999). HnRNP A2 binds to a specific cis-acting element, hnRNP A2 response element (A2RE), found in several dendritically localized mRNAs Munro et al., 1999). This interaction is necessary for transport of A2RE-mRNAs to the periphery of neural cells (Munro et al., 1999;Shan et al., 2003).A2RE-mRNAs, hnRNP A2, and other components form complexes that look by light microscopy like granules that are transported to the cell periphery by a microtubule-and kinesin-based mechanism. In cultured oligodendrocytes and neurons, granules containing A2RE-mRNAs and hnRNP A2 are associated with the cytoskeleton (CSK) . HnRNP A2 and some A2RE-mRNAs copurify with the CSK from rat brain (Hoek et al., 1998;Boccaccio et al., 1999).To identify molecular partners of hnRNP A2, we performed yeast two-hybrid analysis. One of the components identified is the tumor overexpressed gene (TOG) protein, a microtubule-associated protein (MAP) ubiquitously expressed in human tissues. TOG is a single copy gene that generates two mRNA splice variants (Nagase et al., 1995;Charrasse et al., 1998) encoding proteins that differ by a 60-aa insert near the C terminus. In dividing cells, TOG localizes with centrosome and spindle microtubules and may be necessary for microtubule rearrangements and spindle assembly (Charrasse et al., 1998;Lee et al., 2001). Orthologues of colonic and hepatic tumor overexpressed gene (ch-TOG) (Homo sapiens), Dis1p, Stu2p, XMAP215, ZYG-9, and mini spindles, in fission yeast, budding yeast, Xenopus laevis, Caenorhabditis elegans, and Drosophila melanogaster, respectively, have similar functions. TOG and its orthologues all contain multiple HEAT repeats that may serv...

Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway

“…Linkage combines multiple A2RE RNAs into composite granules. This may be accomplished by TOG protein, a granule component that contains multiple binding sites for hnRNP A2 and may function as a multivalent scaffold for coassembly of multiple A2RE RNAs bound to hnRNP A2 molecules into granules (Kosturko et al, 2005;Carson et al, 2006). In postmitotic cells such as neurons, TOG is restricted to the cytoplasm so linkage presumably occurs in the cytoplasm rather than in the nucleus.…”

“…The rate-limiting component in the A2 pathway may not be hnRNP A2 itself, which is present in very high concentrations in most cells (Kosturko et al, 2006). A more likely possibility is TOG protein, which is present in lower concentrations in the cytoplasm where it binds to hnRNP A2 and is a component of RNA granules (Kosturko et al, 2005).…”

The Microtubule-Associated Protein Tumor Overexpressed Gene/Cytoskeleton-Associated Protein 5 Is Necessary for Myelin Basic Protein Expression in Oligodendrocytes

“…Because TOG is associated with MBP mRNA transport granules (Kosturko et al, 2005), the effect of TOG knockdown on the MBP mRNA trafficking pathway was analyzed. MBP expression is developmentally regulated in cultured oligodendrocytes.…”

“…The trafficking complexes termed RNA granules move along microtubules and contain among other components, hnRNP A2 and the microtubule-associated protein tumor overexpressed gene (TOG) . hnRNP A2 and TOG interact in vitro and in vivo Kosturko et al, 2005). MBP mRNA also contains within its 3Ј UTR a cytoplasmic polyadenylation element (CPE) that binds the cytoplasmic polyadenylation element binding protein (CPEB).…”

Characterization of hnRNP A3 isoforms in The A2RE-proteome

“…conventional kinesin and cytoplasmic dynein) (Carson, Worboys, Ainger, & Barbarese, 1997), and linking molecules (e.g. tumor over-expressed gene protein) (Kosturko, Maggipinto, D'Sa, Carson, & Barbarese, 2005).…”