Characterization of ribosome stalling and no-go mRNA decay stimulated by the fragile X protein, FMRP

Abstract

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal non-canonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not poly(A)-binding protein, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of 4 putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes but only stimulates NGD of a small select set of transcripts, revealing a minor role of FMRP that would be misregulated in FXS.