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We thank W. matrix columns (Micro Bio-Spin Columns P-30 Tris, RNase-free; Bio-Rad, Cambridge, MA), and an aliquot of each was subjected to electrophoresis on an agarose-formaldehyde gel to verify purity and estimate the RNA concentration. In real-time experiments, the RNA (1.0 mg/ml) was mixed with Texas Red-conjugated dextran (10 kDa; Molecular Probes) and filtered through an 0.2 m spin filter (Millipore, Bedford, MA) just before microinjection. In all other experiments, the RNA was diluted with DEPC-treated water to a final concentration at 0.5 mg/ml and then filtered through an 0.2 m filter before injection. for 15 min at 4C, the supernatant proteins were resolved by SDS-PAGE and then electroblotted onto Immobilon polyvinylidene difluoride membrane (Millipore, Sydney, Australia). The blots were developed with a mouse antibody to hnRNP A2 (as above; used at a 1:2000 dilution) or with rabbit antibody to -tubulin (used at 1:2000 dilution) as a control for protein loading, followed by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection. For immunofluorescence microscopy, the neurons were fixed and treated with the hnRNP A2 antibody, followed by a fluorescein isothiocyanate-conjugated goat anti-mouse IgG. (this Figure) and Figure 2. For each RNA, at least 40 cells were imaged for analysis. The error bars represent the SDs derived from the binomial distribution. Open in a separate window Figure 4. Transport of A2RE-containing RNAs visualized in live neurons. Hippocampal neurons were microinjected with fluorescently labeled RNA (green) and Texas Red-labeled 10 kDa dextran (red), and after 30 min their distribution was determined by dual-channel confocal microscopy. transcribed RNA, and is not relevant to the current study. Antibodies to hnRNP A2 and antisense oligonucleotides interfere with trafficking Coinjection of A2RE-containing RNA with a monoclonal antibody to hnRNP A2 resulted in reduced trafficking (Fig. 5). In multiple experiments, RNA containing the 21 nt A2RE, coinjected with water or antibodies to irrelevant proteins, was transported in 70% or more of the neurons, in accord with our other experiments on neurons (Fig. 4were similar, as assessed by MAP2 immunofluorescence. Antisense treatment, but not sense treatment, affected the trafficking of RNA in the hippocampal neurons. RNAs containing A2RE, A2RE11, or the MAP2A A2RE-like sequence were transported in sense-treated cells but to a lower extent in antisense-treated cells (Figs. ?(Figs.4used for each of the analyses. Colchicine eliminates RNA trafficking Previous work showed that cellular exposure to the microtubule-disrupting agents nocodazole or colchicine inhibited RNA localization in oligodendrocytes (Carson et al., 1997). Hippocampal neurons treated with colchicine for 30 min did not transport microinjected A2RE-containing RNA and had decreased numbers of hnRNP A2 granules in the processes, yet the overall cell morphology appeared normal (Fig. 8); hnRNP A2-containing granules are evident (arrows, top right) but they do not contain microinjected A2RE RNA (arrows, top left). By contrast, disruption of microfilaments with cytochalasin had no effect on RNA trafficking or hnRNP A2 distribution. Thus A2RE-hnRNP A2 granule trafficking in neurons is dependent on the presence of intact microtubules but independent of microfilament organization. Calcipotriol monohydrate This contrasts with -actin RNA localization in fibroblasts, which is Mouse monoclonal to HER-2 dependent on microfilaments (Sundell and Singer, 1991) but parallels the trafficking of this mRNA in microtubule-associated granules in neurons (Bassell et al., 1998). Open in a separate window Figure 8. Effects of disruption of microtubules on A2RE RNA trafficking. were A8G and G9A. Oligonucleotides with these mutations were found not to bind human recombinant or rat brain hnRNP A2 in biosensor, pull-down, and electrophoretic mobility shift experiments, and these point mutations within a long RNA molecule sufficed to eliminate RNA transport in oligodendrocytes (Munro et al., 1999) and neurons (Figs. ?(Figs.3,3, ?,4).4). Antisense oligonucleotide treatment of neurons resulted in concentration of hnRNP A2 in the nucleus, leaving little in the neurites. By contrast, in Calcipotriol monohydrate oligodendrocytes, treatment with an equivalent oligonucleotide markedly reduced the levels of hnRNP A2 throughout these cells, perhaps reflecting a higher rate of export of this protein to the cytoplasm where it may be degraded and not replaced in the antisense-treated cells. In both cell types, however, antisense Calcipotriol monohydrate treatment compromised A2RE-dependent RNA trafficking. These antisense experiments were supported by others in which the population of cells transporting A2RE-containing RNA into the dendrites was diminished by injection of antibodies to hnRNP along with the RNA, presumably by reduction of the amount of hnRNP A2 available.