Sorting of RNAs to specific cellular loci occurs in diverse settings from fly oocytes to mammalian neurons. Localization of an mRNA is an efficient means to target proteins to specific regions, and thereby enhance specific interactions. Using the membrane permeable nucleic acid stain, SYTO 14, we can directly visualize the translocation of endogenous RNA in living cells. Labeled RNA is nonrandomly distributed as discrete granules in neuronal processes. The labeled granules colocalized with poly(A) mRNA, with the 605 ribosomal subunit, and with elongation factor 1 alpha, suggesting that granules represent a translational unit. A subset of labeled granules co-localized with b-actin mRNA. Correlative light and electron microscopy indicated that the fluorescent granules likely corresponded to clusters of ribosomes at the ultrastructural level. Granules appeared as tight circular clusters of ribosomes. Post-staining of sections with heavy metals confirmed the presence of ribosomes within these granules. This work was performed at the NCMIR using the confocal microscope and the IVEM. In living neurons, a sub-population of RNA granules were motile during the observation period. They moved at an average rate of 0.1 ~m/sec. In young cultures their movements were exclusively anterograde, but after seven days in culture, half of the motile granules moved in the retrograde direction. Granules in neurites were delocalized after treatment with microtubule disrupting drugs. These results raise the possibility of a cellular traificking system for the targeting of RNA in neurons. A manuscript detailing this work has been submitted.
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