The molecular biology of FMRP: new insights into fragile X syndrome

Abstract

Fragile X mental retardation protein (FMRP) is the product of the fragile X mental retardation 1 gene (FMR1), a gene that - when epigenetically inactivated by a triplet nucleotide repeat expansion - causes the neurodevelopmental disorder fragile X syndrome (FXS). FMRP is a widely expressed RNA-binding protein with activity that is essential for proper synaptic plasticity and architecture, aspects of neural function that are known to go awry in FXS. Although the neurophysiology of FXS has been described in remarkable detail, research focusing on the molecular biology of FMRP has only scratched the surface. For more than two decades, FMRP has been well established as a translational repressor; however, recent whole transcriptome and translatome analyses in mouse and human models of FXS have shown that FMRP is involved in the regulation of nearly all aspects of gene expression. The emerging mechanistic details of the mechanisms by which FMRP regulates gene expression may offer ways to design new therapies for FXS.

Figure 1.. Brain cell types affected in FXS.

FXS is a neurodevelopmental disorder resulting from FMRP deficiency, which adversely affects nearly every aspect brain development and functioning of many brain cell types. FMRP-deficient neural stem (progenitor) cells (NSCs) exhibit altered proliferation and fate specification in both developing brains as well as adult neurogenic zones. FMRP-deficient neurons display stunted dendritic morphogenesis, altered axonal targeting, reduced synaptogenesis, and abnormal circuit integration. FMRP is also expressed in astrocytes where it regulates astrocyte-neuron interactions. FMRP deficiency may lead to transient impairment in white matter development.

Figure 2.. Functions of FMRP targets.

FMRP protein contains multiple domains that are important for its functions, including two Agenet/Tudor domains (Agn) for binding DNA and other proteins, nuclear localization (NLS) and exit (NES) sequences, and several RNA binding domains, KH0, KH1, KH2, and RGG box. FMRP binds >1000 mRNAs in the brain that are involved in neural processes such as synaptogenesis (synaptic proteins), cell-cell communication (neurotransmitter receptors), cytoskeleton and microtubule modulators, and metabolic regulators. FMRP also binds to mRNAs encoding transcription factors and epigenetic chromatin modulators, especially in immature cells (e.g. NSCs and immature neurons), although transcription factors and chromatin modulators are not among the top categories identified in neurons or brain tissue.

Figure 3.. Methods for the analysis of transcriptome-wide translation.

TRAP (translating ribosome affinity purification)-seq. Cells or tissues are transduced with an epitope-tagged ribosomal protein (green), which can be expressed in specific cells or tissues (step 1). The cells/tissues are then subjected to immunoprecipitation with antibody against the epitope (usually HA, FLAG, or GFP) (step 2); the co-precipitating RNA is then de-proteinized and sequenced (blue line) (step 3). Note that mRNAs associated with few or no ribosomes are not immunoprecipitted (red line). TRAP-seq has been performed in WT and Fmr1-deficient mouse brain^, and has identified mRNAs under translational control by FMRP in specific neuron subtypes. Steady state ribosome profiling. In cells or tissues, ribosomes are “frozen” on RNA by treatment with cycloheximide (step 1). The RNA is digested with RNase (step 2) and the small mRNA fragments protected from hydrolysis by the ribosomes are collected and sequenced (step 3). Input total RNA, which serves as a measure of all mRNA in the cells is also sequenced (step 4). The ribosome protected fragments (RPFs) and input mRNA are then aligned to the reference genome (step 5). RPFs are generally higher of the start (AUG) and stop (TAA) codons and are not present on 5’ or 3’ untranslated regions. Steady state ribosome profiling from WT and Fmr1-deficient cells or brain tissue has identified mRNAs whose translation is up and down regulated by FMRP^,,,. Dynamic ribosome profiling. Cells or tissues are treated with homoherringtonine (HHT), which freezes ribosomes on initiation codons but allows translocating ribosomes to continue to elongate polypeptides (step 1). The ribosomes are evident at t₁₀ and t₂₀ when compared to t₀. At various time points after HHT, the translocating ribosomes are frozen by treatment with cycloheximide (step 2), which is followed by ribosome profiling, as described above (step 3). Input RNA is also sequenced (step 4). Because there are fewer ribosomes at, for example, t₂₀ relative to t₀, there are fewer ribosome footprints. Ribosome footprints on the AUG start codon (green) are maintained throughout the time course where as footprints on the TAA stop codon (red) are generally reduced over time (step 5). The time it takes for ribosomes runoff following HHT treatment has demonstrated that on most mRNAs, ribosomes translocate quickly whereas on other mRNAs, the ribosomes translocate slowly or nearly not at all. Dynamic ribosome profiling performed using WT and Fmr1-deficient mouse brain has revealed that FMRP stalls ribosomes on specific mRNAs. Note that dynamic ribosome profiling, but not steady state ribosome profiling, can distinguish between translocating and stalled ribosomes.

Figure 4.. Mechanisms of FMRP-regulated translation.

A. FMRP as a roadblock to ribosome translocation. FMRP binding to mRNA might impede ribosome translocation, thereby leading in reduced polypeptide elongation. B. FMRP as a ribosomal protein that inhibits tRNA association with the ribosome. In vitro reconstitution experiments followed by cryo-EM analysis suggest that FMRP binds the 60S ribosomal subunit in such a manner as to prevent tRNA and/or elongation assembly into the ribosome, thereby causing ribosome stalling C. FMRP and codon optimality. According to one current model, FMRP directly or indirectly associates with the translational machinery on wild type mouse cortex mRNAs that have optimal codons and stalls ribosomes (as indicated by the blue |-). In this model, FMRP also blocks a hypothetical nuclease(s) (purple pac-man) to prevent RNA degradation. Because there is no FMRP association with the translational machinery on RNAs with nonoptimal codons, these RNAs have normal ribosome translocation but the mRNAs are unstable because nuclease attack is not hindered by FMRP. Because of this RNA instability, there is reduced protein production. In FMRP-deficient cortex, RNAs with optimal codons are associated with normally translocating ribosomes, but the RNAs are unstable because there is no FMRP to block prevent nuclease hydrolysis. RNAs with nonoptimal codons behave similarly in FMRP-deficient cortex as they do in wild type cortex. According to this model, FMRP regulates protein synthesis primarily at the level of RNA degradation as opposed to translational control.

Figure 5.. Summary of FMRP activities.

FMRP is a multi-functional protein with diverse mechanisms of action; it binds mRNAs and may regulate their translation, stability, editing, or intracellular transport. FMRP also directly interacts with proteins (such as ion channels) and regulates their function. Finally, FMRP can regulate RNA synthesis by either controlling the expression of or modulating the activities of transcription factors and chromatin modifying enzymes