Regulation of FOSB MRNA Isoforms by Drugs of Abuse

ΔFosB, a truncated splice isoform of FosB, is a transcription factor that accumulates within a subset of neurons after chronic administration of drugs of abuse or other chronic stimuli. Due likely to its structure and post-translational modifications, ΔFosB protein is uniquely stable relative to the transiently expressed full-length FosB and all other Fos family proteins. The goal of this study was to determine if the relative expression of the two fosB isoforms is regulated at the mRNA level, thereby further contributing to the accumulation of ΔFosB. First, unlike the protein, the half-life of ΔfosB mRNA is only slightly longer than that of full-length fosB mRNA both in cultured cells in vitro and in the brain in vivo. Additionally, similar to c-fos, both fosB isoforms are induced abundantly in striatum after acute administration of amphetamine and partially desensitize after chronic dosing. Surprisingly, the relative ratio of the fosB to ΔfosB mRNA (normally 16:1 in saline controls) decreases significantly only after acute doses or at doses that elicit the greatest induction of both transcripts. When acute amphetamine doses are incrementally increased, fosB levels are induced to roughly equivalent levels regardless of dose; however, ΔfosB levels increase as the drug dose increases. A similar pattern of fosB and ΔfosB mRNA induction was seen in cell culture. These findings suggest that the splicing of fosB RNA may be regulated by the quantity of unspliced transcript available to the splicing machinery. That is, above a certain threshold of full-length fosB, the remaining primary transcript is alternatively spliced into ΔfosB. This splicing phenomenon is likely regulated by the Polypyrimidine Tract Binding (PTB1) protein. PTB1 protein has been shown to inhibit the U2AF splicing complex and thus prevent alternative splicing of regions in close proximity to where it is bound. It has previously been demonstrated that PTB1 protein binds the fosB transcript in vitro. Here, it is shown that overexpression of PTB1 in PC12 cells alters the ratios of the fosB isoforms by increasing the amount of fosB transcript relative to ΔfosB transcript. Therefore, this study concludes that under basal conditions PTB1 protein binds the majority of the fosB premRNA, thereby inhibiting the generation of the ΔfosB transcript. Only when PTB1 protein is saturated with transcript does the ratio of fosB to ΔfosB decrease significantly, because the unbound pre-mRNA is spliced into ΔfosB. These data provide fundamental information concerning the generation of ΔfosB mRNA and indicate the selective accumulation of ΔFosB protein with chronic drug exposure does not involve its preferential generation by splicing.