RNA-Binding Specificity of the Human Fragile X Mental Retardation Protein

Athar, Youssi Momen; Joseph, Simpson

doi:10.1016/j.jmb.2020.04.021

Cited by 21 publications

(44 citation statements)

References 73 publications

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“…Two recent studies found specific rRNA expansion segments in the ribosomes of higher eukaryotes contain G-quadruplex structures [37,38]. Together with our recent study showing the RGG domain of hFMRP can bind even a minimal G-quadruplex structure, we investigated whether the RGG-CTD region of hFMRP could be binding directly to a G-quadruplex on the ribosome to inhibit translation [24]. To determine whether binding to the ribosome is correlated with translation inhibition, we tested the binding of the human 80S ribosome to NT-hFMRP, GST-hFMRP RGG, and GST-hFMRP KH0-KH1-KH2 by sucrose cushion co-sedimentation.…”

Section: Fmrp Binding To the Ribosome Correlates With Inhibition Of Tmentioning

confidence: 93%

“…It is possible that the KH domains of FMRP bind to pseudoknot structures in the mRNAs; however, such structures are not common in mRNAs and do not agree with the fact that FMRP appears to interact with thousands of mRNAs. In contrast, the RGG domain of FMRP binds to GQ structures with high affinity, and potential GQ structures are prevalent in mRNAs [24,[30][31][32][33][34][35][36]. Interestingly, GQ structures are even present in the 28S and 18S rRNA expansion segments [37,38].…”

Section: Introductionmentioning

confidence: 99%

“…Based on these results, it was proposed that FMRP may bind to mRNAs that possess GQ or pseudoknot forming sequences to repress their translation [12,20,22,23]. We previously showed that the KH domains of FMRP do not bind to simple four or five nucleotide RNA sequence motifs compared to the canonical KH domains found in other RNA-binding proteins [24][25][26][27][28][29]. It is possible that the KH domains of FMRP bind to pseudoknot structures in the mRNAs; however, such structures are not common in mRNAs and do not agree with the fact that FMRP appears to interact with thousands of mRNAs.…”

Section: Introductionmentioning

confidence: 99%

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The Human Fragile X Mental Retardation Protein Inhibits the Elongation Step of Translation through its RGG and C-terminal domains

Athar¹,

Joseph²

2020

Preprint

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Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates the translation of numerous mRNAs in neurons. The precise mechanism of translational regulation by FMRP is unknown. Some studies have indicated that FMRP inhibits the initiation step of translation, whereas other studies have indicated that the elongation step of translation is inhibited by FMRP. To determine whether FMRP inhibits the initiation or the elongation step of protein synthesis, we investigated m7G-cap-dependent and IRES-driven, cap-independent translation of several reporter mRNAs in vitro. Our results show that FMRP inhibits both m7G-cap-dependent and cap-independent translation to similar degrees, indicating that the elongation step of translation is inhibited by FMRP. Additionally, we dissected the RNA-binding domains of hFMRP to determine the essential domains for inhibiting translation. We show that the RGG domain, together with the C-terminal domain (CTD), is sufficient to inhibit translation while the KH domains do not inhibit mRNA translation. However, the region between the RGG domain and the KH2 domain may contribute as NT-hFMRP shows more potent inhibition than the RGG-CTD tail alone. Interestingly, we see a correlation between ribosome binding and translation inhibition, suggesting the RGG-CTD tail of hFMRP may anchor FMRP to the ribosome during translation inhibition.

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Section: Fmrp Binding To the Ribosome Correlates With Inhibition Of Tmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Human Fragile X Mental Retardation Protein Inhibits the Elongation Step of Translation through its RGG and C-terminal domains

Athar¹,

Joseph²

2020

Preprint

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“…The functionality of the purified proteins was verified by testing the FXP family's binding to a G-quadruplex forming RNA, a well-known target of FMRP, which is bound by the RGG motif, and a target of FXR1P 11,30,35,36,41,54 . We chose to test the proteins' binding to poly-G17U as our lab has identified poly-G17U as a G-quadruplex forming RNA 55 . For a negative control we used CR1, an RNA with no predicted G-quadruplex forming capability.…”

Section: Analysis Of the Rna-binding Activity Of The Fragile X Proteinsmentioning

confidence: 99%

“…Samples were excited at 485 nm and emission was measured at 535 nm. To determine binding affinities, the anisotropy data from each binding assay were normalized to initial values without protein, plotted, and fit to a quadratic equation as previously described 55 . Three independent trials were performed (except for FMRP 1 and 2 nM points) to determine standard deviations.…”

Section: G-quadruplex Rna Binding Of the Fragile X Proteinsmentioning

confidence: 99%

A Simple Procedure for Bacterial Expression and Purification of the Fragile X Protein Family

Edwards

Joseph

2020

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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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