G-quartet-dependent recognition between the FMRP RGG box and RNA

Ramos, Andrés; Hollingworth, David; Pastore, Annalisa

doi:10.1261/rna.5960503

Cited by 118 publications

(121 citation statements)

References 35 publications

(41 reference statements)

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“…In accordance with data from our previous study (6), we found that the N terminus of ICP27 is flexible and not well folded. NMR analysis of the RGG box region of FMRP also showed that this region was flexible and unstructured; however, when a Gquartet sequence was present, the FMRP RGG box region became more structured (30). In the case of ICP27, we did not discern any differences in the spectra of the N-terminal protein alone compared to those of the N-terminal protein in the presence of gC sequences to which it binds (Fig.…”

Section: Discussionmentioning

confidence: 65%

“…Because our previous results showed that the N-terminal 160 amino acids of ICP27 are highly flexible (6), we sought to determine if ICP27 bound to a gC oligonucleotide would confer more rigidity or folding on the N terminus. This was seen in NMR analyses of the cellular protein fragile X mental retardation protein (FMRP) when the RGG box was bound to G-quartet RNA substrates (30). The protein encoding the N-terminal 160 amino acids of ICP27 was expressed in E. coli Rosetta cells in minimal medium containing 15 NH 4 Cl to isotopically label the expressed proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we showed that ICP27 binds specifically to GC-rich sequences but that the N-terminal portion of ICP27 encoding the RGG box does not bind Gquartet structures (5). This finding was surprising because the RGG boxes of two other proteins that bind nucleic acids, FMRP and EBNA1, have been shown to bind to G-quadruplex or G-quartet structures (29,30). G quartets are complex tertiary structures that can form readily in DNA or RNA containing three or more consecutive guanines (4,8,30).…”

Section: Discussionmentioning

confidence: 99%

“…This finding was surprising because the RGG boxes of two other proteins that bind nucleic acids, FMRP and EBNA1, have been shown to bind to G-quadruplex or G-quartet structures (29,30). G quartets are complex tertiary structures that can form readily in DNA or RNA containing three or more consecutive guanines (4,8,30). Not only does the N-terminal 160 amino acids of ICP27, containing the RGG box, not bind to G-quartet structures, but NMR analysis of the sequences to which it does bind showed that ICP27 also does not bind to highly structured sequences but binds only to sequences that are flexible (5).…”

Section: Discussionmentioning

confidence: 99%

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Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Corbin-Lickfett¹,

Souki²,

Cocco

et al. 2010

J Virol

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ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A) ؉ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral infection. ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2,3,5,10,13,21,34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20,21). ICP27 also interacts with the export adaptor proteins Aly/REF (2,3,23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5Ј end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11,36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16,17,27) by binding RNA and interacting with TAP/ NXF1 (14,16,18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of new...

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Section: Discussionmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Corbin-Lickfett¹,

Souki²,

Cocco

et al. 2010

J Virol

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“…Some of these RNAs harbor motifs that may form G-quartets and G-quadruplexes in vivo (21, 25). Several mRNAs contain regions shown to form G-quadruplexes implicated in FMRP binding in vitro (21,24,(26)(27)(28)(29). Notably, binding of FMRP to G-rich RNAs in vitro requires only the RGG motif, which specifically interacts with natural and in vitro selected G-quadruplex-containing RNAs such as a 35-nucleotide sc1 RNA (21,(26)(27)(28)(29).…”

mentioning

confidence: 99%

Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

Vasilyev

Polonskaia

Darnell

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

177

181

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Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-Å crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K + -stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex-quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation-π interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I β-turn, a secondary structure element whose formation was not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex-quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG-RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.R NA-binding proteins (RBPs) control all aspects of RNA metabolism and are fundamental to core cellular processes. ∼14% of identified human RBPs are implicated in a broad spectrum of human pathologies, including neurodegenerative and muscular diseases, metabolic disorders, and cancers (1, 2). Fragile X Mental Retardation Protein (FMRP) is among the most important RBPs because of its central role in several human diseases (3). Loss of FMRP function due to CGG triplet repeat expansion-associated transcriptional silencing or missense mutations in the protein (4, 5) lead to fragile X syndrome (FXS), the most common cause of inherited intellectual disability. Mutations in FMRP are also the leading monogenic cause of autism (6, 7). Intermediate length repeat expansions in the FMR1 gene are linked to fragile X-associated tremor ataxia syndrome (8) and fragile X-associated primary ovarian insufficiency (9).FMRP contains four canonical nucleic acid-binding motifs, three KH domains and one arginine-glycine-rich (RGG) box, which mediate interactions with RNAs in mRNA transport, storage, stability, and regulation of translation (Fig. 1A) (3, 10). Each KH domain has been reported to have a mutation either causing FXS or found in patients with intellectual disability (4, 5, 11). In neurons, FMRP associates with a subset of mRNAs and represses their translation both in the cell body and near synapses (12). Loss of repression of these mRNAs is associated with alterations in synaptic plasticity and dendritic spine dynamics thought to underlie...

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Joining the dots – protein‐RNA interactions mediating local mRNA translation in neurons

Gallagher

Ramos

2018

FEBS Letters

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Establishing and maintaining the complex network of connections required for neuronal communication requires the transport and in situ translation of large groups of mRNAs to create local proteomes. In this Review, we discuss the regulation of local mRNA translation in neurons and the RNA-binding proteins that recognise RNA zipcode elements and connect the mRNAs to the cellular transport networks, as well as regulate their translation control. However, mRNA recognition by the regulatory proteins is mediated by the combinatorial action of multiple RNA-binding domains. This increases the specificity and affinity of the interaction, while allowing the protein to recognise a diverse set of targets and mediate a range of mechanisms for translational regulation. The structural and molecular understanding of the interactions can be used together with novel microscopy and transcriptome-wide data to build a mechanistic framework for the regulation of local mRNA translation.

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G-quartet-dependent recognition between the FMRP RGG box and RNA

Cited by 118 publications

References 35 publications

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

Joining the dots – protein‐RNA interactions mediating local mRNA translation in neurons

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