Akins, M. R. | Search bioRxiv

bioRxiv2018

The FXR2P low complexity domain drives assembly of multiple fibril types with differing ribosome association in neurons

RNA binding proteins (RBPs) typically function in higher order assemblages to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of Fragile X granules, which associate with axonal mRNA and ribosomes in the intact brain. Here we performed an unbiased EGFP insertional mutagenesis screen to probe for FXR2P domains important for assembly into higher order structural states in neurons. Fifteen of the 18 unique in-frame FXR2PEGFP fusions tested formed cytosolic granules. However, EGFP insertion within a 23 amino acid region of the low complexity (LC) domain induced formation of distinct FXR2PEGFP fibrils (A and B) that were found in isolation or assembled into highly ordered bundles. Type A and B complexes exhibited different developmental timelines, ultrastructure and ribosome association with ribosomes absent from bundled Type B fibrils. The formation of both fibril types was dependent on an intact RNA binding domain. We conclude that formation of these higher order FXR2P assemblages with alternative structural and compositional states in neurons requires collaboration between the LC and RNA binding domains.\n\nSummary StatementThe Fragile X protein FXR2P forms multiple types of fibrillar assemblages with differential ribosome associations in neurons through cooperation between its RNA binding and LC domains.

neuroscience↗

bioRxiv2018

FMRP association with and regulation of Fragile X granules exhibit circuit-dependent requirements for the KH2 RNA binding domain

The localization and translation of mRNAs is controlled by a diverse array of ribonucleoprotein particles (RNPs), multimolecular complexes containing mRNAs and RNA binding proteins. Fragile X granules (FXGs) are a family of RNPs that exemplify the diversity of RNA granules in the mammalian nervous system. FXGs are found in a conserved subset of neurons, where they localize exclusively to the axonal compartment. Notably, the specific RNA binding proteins and mRNAs found in FXGs depend on brain circuit and neuron type, with all forebrain FXGs containing Fragile X mental retardation protein (FMRP), the protein mutated in the human autism-related disorder Fragile X syndrome. FMRP negatively regulates FXG abundance but is not required for their association with ribosomes or mRNA. To better understand the circuit-dependent mechanisms whereby FMRP associates with and regulates FXGs, we asked how a disease-causing point mutation, I304N, in the KH2 RNA binding domain of FMRP affects these granules in two brain regions - cortex and hippocampus. We found that FMRPI304N had a reduced association with FXGs, as it was absent from approximately half of FXGs in cortex and nearly all FXGs in hippocampus. FXG abundance correlated with the number of FMRP-containing FXGs, suggesting that FMRP regulates FXG abundance by KH2-independent mechanisms that occur locally within the granules. Together, these findings illustrate that cell type-dependent mechanisms guide the assembly of similar RNA granules. Further, point mutations in RNA granule components may lead to cell type-dependent phenotypes that produce atypical forms of disorders that normally arise from more severe mutations.

neuroscience↗