Elevated FMR1 mRNA in premutation carriers is due to increased transcription

Abstract

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.

FIGURE 1.

Diagram of the approximate locations of primers and probes used for the current RT-PCR analysis. Sequences of the primers and probes are given in Table 1.

FIGURE 2.

Relative FMR1 mRNA and transcript levels in premutation versus normal lymphoblastoid cell lines. (A) Analysis of spliced message using primers located in exons 3 and 4, with the probe spanning the intron/exon junction. (B) Analysis of pre- or partially spliced transcripts using primers located in introns 2 and 3 (MM₁) or in exon 3 and intron 4 (MM₂). GM, hypermethylated, full mutation allele (∼600 CGG repeats); AG, normal allele (female; 16, 29 CGG repeats); MM, large premutation allele (160 CGG repeats). Error bars reflect measurements performed in triplicate.

FIGURE 3.

In situ hybridization with β-tubulin and FMR1 antisense strand in lymphoblastoid cell line derived from a normal individual (AG), from a male premutation carrier (TF, 190 CGG repeats), and from an individual with a hypermethylated full mutation (GM, ∼600 CGG repeats).

FIGURE 4.

Expansion of the CGG repeat in the FMR1 mRNA does not affect the fraction of message that possesses a 5′ cap. Capped and uncapped mRNAs were fractionated by affinity chromatography (see Materials and Methods); capped and uncapped mRNAs were quantified by TaqMan RT-PCR, using either GUS (A) or GAPDH (B) mRNAs as reference genes.

FIGURE 5.

Graph of the relationship between relative FMR1 mRNA levels, the number of CGG repeats, and the number of AGG interruptions, for a cohort of 128 adult male premutation carriers and controls. Gray circles, 2 AGG interruptions; open circles, 1 AGG; black circles, 0 AGGs. (Inset) Histogram representing the average FMR1 mRNA levels for premutation alleles with 0 AGG interruptions or with 1 AGG interruption (includes two cases with 2 AGGs); error bars represent SEMs.