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

Local protein synthesis is a crucial process that maintains synaptic proteostasis. Ribosomes stalled at elongation make up a sizable percentage of mRNAs translated in developing neurons. The protein lost in Fragile X syndrome (FMRP) is highly enriched in RNA granules containing stalled ribosomes, both of which play crucial roles in regulating forms of synaptic plasticity such as metabotropic glutamate receptor-dependent long-term depression (mGLUR-LTD). Previous examination of ribosome protected fragments (RPFs) from neuronal stalled ribosomes have identified motifs matching 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 if FMRP recognition of these sequences is important for determining where mRNAs are stalled, we examined stalled ribosomes RPFs isolated from P5 mice lacking the FMRP protein. We found that the loss of FMRP had no effect on the proteins associated with RNA granules, structure of the ribosomes and stalling sites, or where accumulations of RPFs occurred. However, there was a significant decrease in the amount of stalling on mRNAs previously shown to be associated with FMRP by cross-linking immunoprecipitation (CLIP). In addition, there was a decrease in the number of RNA granules containing stalled polysomes as assayed by ribopuromycylation in distal neurites and, unlike RNA granules in WT neurons, the remaining distal RNA granules were resistant to reactivation by stimuli that induce mGLuR-LTD. These results show important roles for FMRP in regulating RNA granules containing stalled ribosomes, but not in determining where ribosomes are stalled.