No abstract

ABSTRACT. The localization of mRNA in neuronal dendrites plays a role in both locally and temporally regulated protein synthesis, which is required for certain forms of synaptic plasticity. RNA granules constitute a dendritic mRNA transport machinery in neurons, which move along microtubules. RNA granules contain densely packed clusters of ribosomes, but lack some factors that are required for translation, suggesting that they are translationally incompetent. Recently some of the components of RNA granules have been identified, and their functions are in the process of being examined, in attempts to better understand the properties of RNA granules. Mammalian Staufen, a double-stranded RNA binding protein, is a component of RNA granules. Staufen is localized in the somatodendritic domain of neurons, and plays an important role in dendritic mRNA targeting. Recently, one of the mammalian homologs of Staufen, Staufen2 (Stau2), was shown to shuttle between the nucleus and the cytoplasm. This finding suggests the possibility that Stau2 binds RNA in the nucleus and that this ribonucleoprotein particle is transported from the nucleus to RNA granules in the cytoplasm. A closer study of this process might provide a clue to the mechanism by which RNA granules are formed.

The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3.

Tap/NXF1, the founding member of the evolutionarily conserved NXF (Nuclear RNA export Factor) family of proteins, is required for the nuclear export of bulk poly(A)+ RNAs. In mice, three additional NXF family genes (NXF2, NXF3, NXF7) have been identified and characterized to date. Cumulative data suggest that NXF family members play roles, not only in nuclear mRNA export, but also in various aspects of post-transcriptional mRNA metabolism. In order to better understand the functional role of NXF2, we searched for its binding partners by yeast two-hybrid screening and identified several cytoplasmic motor proteins, including KIF17. The interaction of NXF2 with KIF17, which was confirmed by GST pull-down and co-immunoprecipitation assays, is mediated by the N-terminal domain of NXF2, which is required for the punctate localization patterns in dendrites of primary neurons. We also found that the NXF2-containing dendritic granules, which were co-localized with KIF17, mRNA and Staufen1, a known component of neuronal mRNA granules, moved bidirectionally along dendrites in a microtubule-dependent manner. These results suggest that NXF2, a nucleo-cytoplasmic mRNA transporter, plays additional roles in the cytoplasmic localization of mRNAs through interactions with cytoplasmic motor proteins.