Identifying intrinsic and extrinsic determinants that regulate internal initiation of translation mediated by the FMRI 5′ leader

Background: Regulating synthesis of the Fragile X gene (FMRI) product, FMRP alters neural plasticity potentially through its role in the microRNA pathway. Cap-dependent translation of the FMRI mRNA, a process requiring ribosomal scanning through the 5' leader, is likely impeded by the extensive secondary structure generated by the high guanosine/cytosine nucleotide content including the CGG triplet nucleotide repeats in the 5' leader. An alternative mechanism to initiate translation - internal initiation often utilizes secondary structure to recruit the translational machinery. Consequently, studies were undertaken to confirm and extend a previous observation that the FMRI 5' leader contains an internal ribosomal entry site (IRES). Results: Cellular transfection of a dicistronic DNA construct containing the FMRI 5' leader inserted into the intercistronic region yielded significant translation of the second cistron, but the FMRI 5' leader was also found to contain a cryptic promoter possibly confounding interpretation of these results. However, transfection of dicistronic and monocistronic RNA ex vivo or in vitro confirmed that the FMRI 5' leader contains an IRES. Moreover, inhibiting cap-dependent translation ex vivo did not affect the expression level of endogenous FMRP indicating a role for IRES-dependent translation of FMRI mRNA. Analysis of the FMRI 5' leader revealed that the CGG repeats and the 5' end of the leader were vital for internal initiation. Functionally, exposure to potassium chloride or intracellular acidification and addition of polyinosinic:polycytidylic acid as mimics of neural activity and double stranded RNA, respectively, differentially affected FMRI IRES activity. Conclusion: Our results indicate that multiple stimuli influence IRES-dependent translation of the FMRI mRNA and suggest a functional role for the CGG nucleotide repeats.