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

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 PABPC1, 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 the 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 and can stimulate NGD in cells, albeit on a small number of transcripts, revealing an unappreciated role of FMRP that would be misregulated in FXS when FMRP is lost. N2A cells were obtained from ATCC (# CCL-131) and maintained in high glucose DMEM (Thermo # 11995065) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin in standard tissue culture-treated plastics at 37°C with 5% CO2. N2A cells were seeded in 12-well plates 24 hrs prior to knockdown, and then transfected with Silencer Select siRNAs (Thermo) using Lipofectamine RNAiMAX (Thermo # 13778150) following the manufacturer’s recommendation. At the time of transfection, N2A cells were at ~20% confluency. 24 hrs post siRNA transfection, the media was changed. After 72 hr transfection, total RNA or protein was harvested by TRIzol.