The fragile X protein family consists of three RNA-binding proteins involved in translational regulation. Fragile X mental retardation protein (FMRP) is well-studied, as its loss leads to fragile X syndrome, a neurodevelopmental disorder which is the most prevalent form of inherited mental retardation and the primary monogenetic cause of autism. Fragile X related proteins 1 and 2 (FXR1P and FXR2P) are autosomal paralogs of FMRP that are involved in promoting muscle development and neural development, respectively. There is great interest in studying this family of proteins, yet researchers have faced much difficulty in expressing and purifying the full-length versions of these proteins in sufficient quantities. We have developed a simple, rapid, and inexpensive procedure that allows for the recombinant expression and purification of full-length human FMRP, FXR1P, and FXR2P from Escherichia coli in high yields, free of protein and nucleic acid contamination. In order to assess the proteins’ function after purification, we confirmed their binding to pseudoknot and G-quadruplex forming RNAs as well as their ability to regulate translation in vitro.
The fragile X proteins (FXPs) are a family of RNA-binding proteins that regulate mRNA translation to promote proper neural development and cognition in mammals. Of particular interest to researchers is the fragile X mental retardation protein (FMRP), as its absence leads to a neurodevelopmental disorder: fragile X syndrome (FXS), the leading monogenetic cause of autism spectrum disorders. A primary focus of research has been to determine mRNA targets of the FXPs in vivo through pull-down techniques, and to validate them through in vitro binding studies; another approach has been to perform in vitro selection experiments to identify RNA sequence and structural targets. These mRNA targets can be further investigated as potential targets for FXS therapeutics. The most established RNA structural target of this family of proteins is the G-quadruplex. In this article, we report a 99 nucleotide RNA target that is bound by all three FXPs with nanomolar equilibrium constants. Furthermore, we determined that the last 102 amino acids of FMRP, which includes the RGG motif, were necessary and sufficient to bind this RNA target. To the best of our knowledge, this is one of only a few examples of non-G-quadruplex, non-homopolymer RNAs bound by the RGG motif/C-termini of FMRP.
All cells use organized lipid compartments to facilitate specific biological functions. Membrane‐bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid‐based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)‐modified phospholipids that form stable covalent linkages with an O6‐methylguanine DNA methyltransferase (SNAP‐tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid‐aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP‐tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid‐based artificial organelles.
The fragile X protein family consists of three RNA-binding proteins involved in translational regulation. Fragile X mental retardation protein (FMRP) is well-studied, as its loss leads to fragile X syndrome, a neurodevelopmental disorder which is the most prevalent form of inherited mental retardation and the primary monogenetic cause of autism. Fragile X related proteins 1 and 2 (FXR1P & FXR2P) are autosomal paralogs of FMRP that are involved in promoting muscle development and neural development, respectively. There is great interest in studying this family of proteins, yet researchers have faced much difficulty in expressing and purifying the full-length versions of these proteins in sufficient quantities. We have developed a simple, rapid, and inexpensive procedure that allows for the recombinant expression and purification of full-length human FMRP, FXR1P, and FXR2P from Escherichia coli in high yields, free of protein and nucleic acid contamination. In order to assess the proteins' function after purification, we confirmed their binding to pseudoknot and G-quadruplex forming RNAs. IntroductionThe fragile X protein (FXP) family consists of three RNA-binding, ribosome-associating proteins involved in translational regulation: fragile X-related protein 1 (FXR1P), fragile X-related protein 2 (FXR2P), and the most well-known, fragile X mental retardation protein (FMRP) 1,2,3,4 .FMRP's role in translation repression has been studied extensively, as loss of FMRP expression results in a neurodevelopmental disorder called fragile X syndrome (FXS), the most prevalent form of inherited intellectual disability, and the primary monogenic cause of autism spectrum disorders 5,6,7 . FXS predominantly results from a CGG trinucleotide repeat expansion in the 5' untranslated region of the FMR1 gene 6,7 . The expanded repeats are hypermethylated causing transcriptional silencing of the FMR1 gene, leading to a deficiency or absence of FMRP 6,7,8,9 .Patients with this disorder may experience seizures, hyperactivity, anxiety, and poor language development 7 . On a cellular level, patients with FXS possess a greater density of dendritic spines, and increased numbers of long and immature-shaped spines 10 . It is estimated that 1/5,000 males and 1/4,000-8,000 females possess the full FXS mutation 7 .While perhaps lesser known, FMRP's autosomal paralogs FXR2P and FXR1P are also of interest for their role in translational regulation 1,2,11 . FXR2P-deficient mice have impaired dendritic maturation of new neurons, with new neurons possessing shorter and less complex dendrites compared to wild-type mice 12 . These mice revealed decreased neural connectivity as new neurons with shorter dendrites connected to fewer presynaptic neurons 12 . Mice deficient in FXR2P displayed atypical gene expression in the brain and altered behavior, such as hyperactivity, reduced sensitivity to heat stimuli, and reduced prepulse inhibition 13,14 .FXR1P is unique among the fragile X proteins in that three (e-g) of the seven isoforms in mice (a-g) show strong...
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