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- Materials and methods
In mammalian neurons, transport and translation of mRNA to individual potentiated synapses is believed to occur via a heterogeneous population of RNA granules. To identify components of Staufen2-containing granules, we used the yeast two-hybrid system. A mouse fetal cDNA library was screened with the N-terminal fragment of Staufen2 as bait. ZFR, a three zinc finger protein, was identified as an interacting protein. Confocal microscopy showed that ZFR, although mainly nuclear, was also found in the somatodendritic compartment of primary hippocampal neurons where it localized as granule-like structures. Co-localization with Staufen2 was observed in several granules. Biochemical analyses (immunoprecipitation, cell fractionation) further confirmed the ZFR/Staufen2 association. ZFR was shown to interact with at least the Staufen262 isoform, but not with Staufen1. ZFR also co-fractionated with ribosomes and Staufen259 and Staufen252 in a sucrose gradient. Interestingly, knockdown expression of ZFR through RNA interference in neurons relocated specifically the Staufen262, but not the Staufen259, isoform to the nucleus. Our results demonstrate that ZFR is a native component of Staufen2-containing granules and likely plays its role during early steps of RNA transport and localization. They also suggest that one of these roles may be linked to Staufen262-containing RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling.
In neurons, although most of the mRNAs are restricted to cell bodies, some are found in the somatodendritic compartment. As synthesis of proteins occurs in dendrites, dendritic localization of a specific subset of mRNAs and their local translation are thought to allow neurite growth and plasticity at sites distant from the cell body and the differential plasticity of each individual dendrite in response to neighbouring cells (Kiebler and DesGroseillers 2000; Steward and Worley 2001; Bassell and Kelic 2004; Huang and Richter 2004; Sutton and Schuman 2005). In dendrites, RNA and/or RNA-binding proteins have a punctuated distribution and their movement can be observed in living neurons using the RNA-binding dye SYTO14 (Knowles et al. 1996). These observations lead to a model in which differential delivery of new mRNAs occurs in motile structures called RNA granules. RNA granules are large clusters of ribosomes to which RNAs, translation factors and other proteins are associated (Krichevsky and Kosik 2001). Evidences suggest that they are translationally incompetent and represent reservoirs of silent RNA, which upon cell stimulation release RNAs for local translation on polyribosomes (Krichevsky and Kosik 2001).
Proteomic approaches have recently been used to identify protein components of RNA granules (Ohashi et al. 2002; Bannai et al. 2004; Brendel et al. 2004; Kanai et al. 2004; Villace et al. 2004) but the role of identified proteins and their interaction within the granules is still unclear. Therefore, the heterogeneous composition of RNA granules and particles, their site of formation, the mechanism of recognition of specific RNAs and the mechanism of their transport/localization are still to be understood. Identification and characterization of mammalian Staufen binding protein(s) should provide more information on the mechanisms of RNA transport in neurons. Using the yeast two-hybrid assay, a first Stau2-binding protein was identified: the zinc finger protein ZFR. Biochemical characterization demonstrated that ZFR is associated with the Stau262-containing RNP complexes, but not with Stau1-containing granules. Down-regulation of ZFR by RNA interference caused the retention of the Stau262 isoform in the nucleus, suggesting that ZFR may play an early role in Stau2-granule formation.