| Expansion of a CGG--repeat element in the 5' untranslated region (5' UTR) of the fragile X mental retardation 1 (FMR1) gene, to between 55 and 200 CGG repeats ("premutation" range), leads to the late--adult--onset neurodegenerative disorder, fragile X--associated tremor/ataxia syndrome (FXTAS). FXTAS is thought to arise through a direct, toxic gain--of--function of the expanded--repeat mRNA. Both the toxicity and diminished translational efficiency of (FMR1) are thought to be a consequence of stable secondary structure of the CGG--repeat region, which has not been well defined. In particular, it is not clear whether G--G mismatches, which occur every third base, do in fact base--pair. It is now demonstrated, by NMR spectroscopy on CGG--repeat RNAs, that both C•G and G∘G basepairs do form within a well--defined hairpin stem; though the latter, non--canonical basepair exists in a dynamic conformational equilibrium.;One striking difference between premutation--length alleles and those in the normal range (<∼40 repeats) is the reduced number/absence of AGG interruptions in larger alleles. Such interruptions, which generally occur every 9--11 repeats in normal alleles, are thought to disrupt the CGG--repeat hairpin structure. However, there is no apparent difference in Tm of hairpins with and without an AGG interruption at physiological Mg 2+ levels, suggesting that AGGs do not substantially affect the intrinsic stability of CGG--repeat hairpins. The AGG interruptions have also been postulated to have a role in the efficiency of (FMR1) translation by breaking up secondary structure and allowing for more efficient ribosomal scanning. To test this hypothesis, the translational efficiencies of CGG-repeat mRNAs with 0--2 AGG interruptions were measured. Findings demonstrated that AGG interruptions have no detectable influence on translational efficiency, which indicates that any AGG--repeat--induced alterations in secondary/tertiary structure, if present, do not involve the rate--limiting step(s) in translational initiation.;Finally, since many mRNAs that utilize alternative forms of translation initiation (i.e. by internal ribosome entry sites or shunting) have long, highly structured 5'UTRs, the mechanism of translational initiation of the (FMR1) mRNA was examined. The results establish that the preponderance of translational initiation occurs via the canonical scanning mechanism, not by alternative mechanisms that circumvent the CGG--repeat element. |
