Association of spermine and 4S RNA during axonal transport in regenerating optic nerves of goldfish

Abstract

Experiments were designed to determine whether polyamines are bound to 4S RNA and then transported axonally along regenerating optic axons of goldfish. In one set of experiments, inhibition of retinal RNA synthesis by intraocular injections of 10 microgram of cordycepin, blocked the axonal transport of both [3H]RNA and [14C]spermidine by about 65%, 6 and 14 days after injection. Intraocular injections of vinblastine, (0.1, 0.5 or 1.0 microgram) an agent which interrupts axonal transport of proteins, had no effect on retinal RNA synthesis nor on the amount of [14C]spermidine incorporated into the TCA-insoluble fraction of retinal extracts. However, the axonal transport of both [3H]RNA and [14C]polyamines was affected in a dose-dependent fashion; the inhibition of both was approximately 80% at the higher dose. Further evidence for an association between axonally transported 4S RNA and polyamines came from experiments in which regenerating optic axons were cut and allowed to degenerate 6 days after injection of [3H]spermidine into the eye. The loss of optic axons from the tectum 7 days after cutting the nerve resulted in an 86% loss of TCA insoluble polyamines, indicating a largely intra-axonal locus. A similar loss of 4S RNA was found in identical experiments following injections of [3H]uridine into the eye. Finally, experiments were performed in which [3H]spermidine was injected into both eyes of 12 fish whose optic nerves had been regenerating for 18 days. Six days later, fish were sacrificed and RNA was extracted from tectal homogenates by hot phenol and ethanol precipitation. The major stable RNA species were separated by SDS-polyacrylamide disc gel electrophoresis and radioactivity was determined by extraction of 2.0 mm gel slices. Results showed co-migration of 3H with 4S RNA optical density peaks, and not with 28S and 18S ribosomal RNA peaks, suggesting that some polyamine-associated radioactivity is bound to axonally transported 4S RNA. When the nature of that radioactivity was determined on an amino acid analyzer, it was found to be present primarily as spermine and not as the injected compound spermidine. The data are consistent with the hypothesis that some spermine is bound to 4S RNA and then axonally transported along regenerating axons of the goldfish optic nerve.