Elevated FMR1 mRNA in premutation carriers is due to increased transcription

Carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene have levels of FMR1 mRNA that are elevated by as much as 10-fold in peripheral blood leukocytes and CNS tissue. The excess expanded-repeat mRNA, per se, is now believed to result in forms of clinical involvement that are largely restricted to premutation carriers, including the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Although evidence to date suggests that the elevated mRNA is not due to increased stability, the basis for the increase is not known. In the current study, we have determined the relative transcriptional activities of premutation and normal FMR1 alleles using a highly sensitive nuclear run-on assay that involves immunocapture of digoxigenin-modified run-on transcripts followed by PCR amplification of the nascent transcripts. Using the nuclear run-on approach, we demonstrate that the rate of run-on synthesis of FMR1 transcripts is increased in premutation alleles. The current run-on assay should be broadly applicable to studies of other genes with promoters of weak to moderate strength. The fraction of capped FMR1 mRNA remains unaltered for premutation transcripts, indicating that elevated message levels are not due to premature escape from the cotranscriptional capping process. We also show that, in contrast to the situation with myotonic dystrophy, there is no net nuclear sequestration of premutation FMR1 mRNA. Finally, we have demonstrated that AGG interruptions within the CGG repeat element do not influence FMR1 mRNA levels.