FMRP Regulates Neuronal RNA Granules Containing Stalled Ribosomes, Not Where Ribosomes Stall

Local protein synthesis is a crucial process that maintains synaptic proteostasis. A large percentage of mRNAs translated in developing neurons are associated with stalled ribosomes. FMRP, the protein lost in Fragile X syndrome, is highly enriched in RNA granules that contain stalled ribosomes. Previous examination of ribosome protected fragments (RPFs) from stalled neuronal ribosomes has identified motifs that match those found in mRNAs associated with FMRP, as recognized by FMRP cross-linking immunoprecipitation (CLIP) (Anadolu et al, 2023, Journal of Neuroscience doi: 10.1523/JNEUROSCI.1002-22.2023). To investigate whether FMRP recognition of these sequences is important for determining where ribosomes are stalled on mRNAs, we examined stalled ribosomes RPFs isolated from P5 mice of both sexes lacking the FMRP protein. We found that the loss of FMRP had no effect on the proteins associated with neuronal stalled ribosomes, the structure of the ribosomes, or the stalling sites (locations where RPFs accumulated). However, we observed a significant decrease in the levelsof mRNAs previously shown to be associated with FMRP by CLIP in stalled ribosomes. Additionally, the number of neuronal RNA granules containing stalled ribosomes, as assayed by ribopuromycylation in distal neurites, decreased. Unlike neuronal RNA granules in WT neurons, the remaining distal neuronal RNA granules were resistant to reactivation. These results highlight important roles of FMRP in regulating neuronal RNA granules that contain stalled ribosomes, though it does not influence where ribosomes are stalled and is not directly involved in stalled ribosome formation.