Poster abstracts
Poster number 40 submitted by MaKenzie Scarpitti
A non-canonical RNA-binding domain of the Fragile X protein, FMRP, elicits mRNA G-quadruplex-independent translational repression
MaKenzie R. Scarpitti (1,2), Julia E. Warrick (1,2), Evelyn L. Yoder (1,2), Michael G. Kearse (1Department of Biological Chemistry and Pharmacology, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract:
Fragile X syndrome is caused by the loss of functional fragile X mental retardation protein (FMRP) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor that blocks elongation and is found on puromycin- and nuclease-resistant polysomes in brain tissue. Consistent with its role in regulating elongation, previous CLIP-seq experiments have identified that nearly all mRNA-bound FMRP is in the coding sequence. However, which RNA-binding elements, including at least two highly structured KH domains and a small unstructured RGG-box motif, are required to elicit mRNA targeting for translational repression remains debated. Previous work has shown that the RGG-box motif has high affinity to RNA G-quadruplexes and that deletion of the RGG-box motif renders FMRP unable to repress translation. Even though G-quadruplexes are nearly absent in coding sequences and are enriched in the UTRs, these data form the foundation of a leading model in the field where FMRP is targeted to the mRNA coding sequence by affinity to G-quadruplexes. Here, we directly test this model in vitro by using recombinant human FMRP and custom nanoLuciferase reporter mRNAs and show that FMRP inhibits translation independent of mRNA G-quadruplexes. Biochemical dissection of human FMRP revealed a non-canonical RNA-binding domain (ncRBD), formed by the RGG-box motif appended to the C-terminal domain, is sufficient to bind all four homopolymeric RNAs, binds heterologous mRNA, and inhibits translation. However, neither the RGG-box motif nor CTD alone binds mRNA or inhibits translation. Through scanning deletion analysis, we further refined the minimal ncRBD that binds heterologous mRNA and inhibits translation. Our data indicate that FMRP binds mRNA more promiscuously than previously appreciated and that local concentrations of mRNAs rather than sequence specificity may drive FMRP-mediated translational repression in neurons.
Keywords: RNA binding protein, Translation elongation, G-quadruplexes