Initiation of Translation of the FMR1 mRNA Occurs Predominantly through 5′-End-Dependent Ribosomal Scanning

The fragile X mental retardation 1 (FMR1) gene contains a CGG repeat within its 5′ untranslated region (5′UTR) that, when expanded to 55–200 CGG repeats (premutation allele), can result in the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome. The CGG repeat is expected to form a highly stable secondary structure that is capable of inhibiting 5′-cap-dependent translation. Paradoxically, translation in vivo is only mildly impaired within the premutation range, suggesting that other modes of translation initiation may be operating. To address this issue, we translated in vitro a set of reporter mRNAs containing between 0 and 99 CGG repeats in either native (FMR1) or unrelated (heterologous) 5′UTR context. The 5′-cap dependence of translation was assessed by inserting a stable hairpin (HP) near the 5′ end of the mRNAs. The results of the current studies indicate that translation initiation of the FMR1 mRNA occurs primarily by scanning, with little evidence of internal ribosome entry or shunting. Additionally, the efficiency of translation initiation depends on transcription start site selection, with the shorter 5′UTR (downstream transcription start site I) translating with greater efficiency compared to the longer mRNA (start site III) for all CGG-repeat elements studied. Lastly, an HP previously shown to block translation gave differing results depending on the 5′UTR context, in one case initiating translation from within the HP.