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1
Bhatter, Nupur, Rajan Iyyappan, and Purusharth I. Rajyaguru. "Characterizing mutations in and genetic interactions of RGG-motif translation repressor Sbp1." Wellcome Open Research 3 (August 22, 2018): 102. http://dx.doi.org/10.12688/wellcomeopenres.14709.1.
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Background: Mechanisms of mRNA fate decisions play an important role in determining if a given mRNA will be translated, stored or degraded upon arrival to cytoplasm. Sbp1 is an important RGG-motif containing protein that is implicated in mRNA fate decisions since it can affect mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In order to understand the amino acids important for translation repression activity of Sbp1 we performed mutational analysis of Sbp1 combined with assessing its genetic interaction with another RGG-motif protein Scd6. We created two classes of point mutations a) in aromatic residues of the RGG-motif and b) in residues reported to be phosphorylated. Method: Sequence alignment was performed to identify aromatic residues to be mutated based on conservation. Site-directed mutagenesis approach was used to create several point mutations in Sbp1 expressed under galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon overexpression. The ability of Sbp1 to affect repression activity of other decapping activators was tested using the same growth assay. Results: Mutation of several aromatic residues in the RGG-motif of Sbp1 led to a weak rescue phenotype. However the phospho-mimetic mutants of Sbp1 did not lead to any kind of growth defect rescue. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect ability of Sbp1 to cause growth defect. On the other hand absence of Sbp1 does not affect ability of Dhh1 and Pat1 to repress translation. Conclusion: Based on our growth assay analysis we conclude that mutated aromatic residues contribute marginally to repression activity of Sbp1 whereas phospho-mimetic mutants do not alter ability of Sbp1 to cause growth defect. Interestingly Scd6 does not affect ability of Sbp1 to repress translation, which in turn does not affect Dhh1 and Pat1.
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Corley, Susan M., and Jill E. Gready. "Identification of the RGG Box Motif in Shadoo: RNA-Binding and Signaling Roles?" Bioinformatics and Biology Insights 2 (January 2008): BBI.S1075. http://dx.doi.org/10.4137/bbi.s1075.
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Using comparative genomics and in-silico analyses, we previously identified a new member of the prion-protein (PrP) family, the gene SPRN, encoding the protein Shadoo (Sho), and suggested its functions might overlap with those of PrP. Extended bioinformatics and conceptual biology studies to elucidate Sho's functions now reveal Sho has a conserved RGG-box motif, a well-known RNA-binding motif characterized in proteins such as FragileX Mental Retardation Protein. We report a systematic comparative analysis of RGG-box containing proteins which highlights the motif's functional versatility and supports the suggestion that Sho plays a dual role in cell signaling and RNA binding in brain. These findings provide a further link to PrP, which has well-characterized RNA-binding properties.
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Yun, Chi Y., and Xiang-Dong Fu. "Conserved Sr Protein Kinase Functions in Nuclear Import and Its Action Is Counteracted by Arginine Methylation in Saccharomyces cerevisiae." Journal of Cell Biology 150, no. 4 (August 21, 2000): 707–18. http://dx.doi.org/10.1083/jcb.150.4.707.
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Mammalian serine and arginine–rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein–specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p–Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p–Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.
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Vasilyev, Nikita, Anna Polonskaia, Jennifer C. Darnell, Robert B. Darnell, Dinshaw J. Patel, and Alexander Serganov. "Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP." Proceedings of the National Academy of Sciences 112, no. 39 (September 15, 2015): E5391—E5400. http://dx.doi.org/10.1073/pnas.1515737112.
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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.
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Bhatter, Nupur, Rajan Iyyappan, Gayatri Mohanan, and Purusharth I. Rajyaguru. "Exploring the role of RRM domains and conserved aromatic residues in RGG motif of eIF4G-binding translation repressor protein Sbp1." Wellcome Open Research 3 (September 17, 2021): 102. http://dx.doi.org/10.12688/wellcomeopenres.14709.3.
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Background: RNA binding proteins play crucial role in determining if a given mRNA will be translated, stored, or degraded. Sbp1 is an RGG-motif containing protein that is implicated in affecting mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In this report, we have assessed the genetic interaction of Sbp1 with decapping activators such as Dhh1, Pat1, and Scd6. We have further analyzed the importance of different domains and specific conserved residues of Sbp1 in its ability to cause over-expression mediated growth defect. Method: Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions were created in Sbp1 expressed under a galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon over-expression. The ability of Sbp1 to affect over-expression mediated growth defect of other decapping activators was tested using growth assay. Live cell imaging was done to study localization of Sbp1 and its RRM-deletion mutants to RNA granules upon glucose starvation. Results: Mutation of several aromatic residues in the RGG-motif and that of the phosphorylation sites in the RRM domain of Sbp1 did not affect the growth defect phenotype. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect the ability of Sbp1 to cause growth defect. Moreover, absence of Sbp1 did not affect the growth defect phenotypes observed upon overexpression of decapping activators Dhh1 and Pat1. Strikingly deletion of both the RRM domains (RRM1 and RRM2) and not the RNP motifs within them compromised the growth defect phenotype. Sbp1 mutant lacking both RRM1 and RRM2 was highly defective in localizing to RNA granules. Conclusion: This study identifies an important role of RRM domains independent of the RNP motif in Sbp1 function.
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Wang, Yi-Chun, Shang-Hsuan Huang, Chien-Ping Chang, and Chuan Li. "Identification and Characterization of Glycine- and Arginine-Rich Motifs in Proteins by a Novel GAR Motif Finder Program." Genes 14, no. 2 (January 27, 2023): 330. http://dx.doi.org/10.3390/genes14020330.
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Glycine- and arginine-rich (GAR) motifs with different combinations of RG/RGG repeats are present in many proteins. The nucleolar rRNA 2′-O-methyltransferase fibrillarin (FBL) contains a conserved long N-terminal GAR domain with more than 10 RGG plus RG repeats separated by specific amino acids, mostly phenylanalines. We developed a GAR motif finder (GMF) program based on the features of the GAR domain of FBL. The G(0,3)-X(0,1)-R-G(1,2)-X(0,5)-G(0,2)-X(0,1)-R-G(1,2) pattern allows the accommodation of extra-long GAR motifs with continuous RG/RGG interrupted by polyglycine or other amino acids. The program has a graphic interface and can easily output the results as .csv and .txt files. We used GMF to show the characteristics of the long GAR domains in FBL and two other nucleolar proteins, nucleolin and GAR1. GMF analyses can illustrate the similarities and also differences between the long GAR domains in the three nucleolar proteins and motifs in other typical RG/RGG-repeat-containing proteins, specifically the FET family members FUS, EWS, and TAF15 in position, motif length, RG/RGG number, and amino acid composition. We also used GMF to analyze the human proteome and focused on the ones with at least 10 RGG plus RG repeats. We showed the classification of the long GAR motifs and their putative correlation with protein/RNA interactions and liquid–liquid phase separation. The GMF algorithm can facilitate further systematic analyses of the GAR motifs in proteins and proteomes.
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Krüger, Timothy, Mario Hofweber, and Susanne Kramer. "SCD6 induces ribonucleoprotein granule formation in trypanosomes in a translation-independent manner, regulated by its Lsm and RGG domains." Molecular Biology of the Cell 24, no. 13 (July 2013): 2098–111. http://dx.doi.org/10.1091/mbc.e13-01-0068.
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Ribonucleoprotein (RNP) granules are cytoplasmic, microscopically visible structures composed of RNA and protein with proposed functions in mRNA decay and storage. Trypanosomes have several types of RNP granules, but lack most of the granule core components identified in yeast and humans. The exception is SCD6/Rap55, which is essential for processing body (P-body) formation. In this study, we analyzed the role of trypanosome SCD6 in RNP granule formation. Upon overexpression, the majority of SCD6 aggregates to multiple granules enriched at the nuclear periphery that recruit both P-body and stress granule proteins, as well as mRNAs. Granule protein composition depends on granule distance to the nucleus. In contrast to findings in yeast and humans, granule formation does not correlate with translational repression and can also take place in the nucleus after nuclear targeting of SCD6. While the SCD6 Lsm domain alone is both necessary and sufficient for granule induction, the RGG motif determines granule type and number: the absence of an intact RGG motif results in the formation of fewer granules that resemble P-bodies. The differences in granule number remain after nuclear targeting, indicating translation-independent functions of the RGG domain. We propose that, in trypanosomes, a local increase in SCD6 concentration may be sufficient to induce granules by recruiting mRNA. Proteins that bind selectively to the RGG and/or Lsm domain of SCD6 could be responsible for regulating granule type and number.
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Bhatter, Nupur, Rajan Iyyappan, and Purusharth I. Rajyaguru. "Exploring the role of RRM domains and conserved aromatic residues in RGG motif of eIF4G-binding translation repressor protein Sbp1." Wellcome Open Research 3 (February 6, 2020): 102. http://dx.doi.org/10.12688/wellcomeopenres.14709.2.
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Background: Mechanisms of mRNA fate decisions play an important role in determining if a given mRNA will be translated, stored or degraded upon arrival to cytoplasm. Sbp1 is an important RGG-motif containing protein that is implicated in affecting mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In this report we have assessed the genetic interaction of Sbp1 with decapping activators such as Dhh1, Pat1 and Scd6. We have further analyzed the importance of different domains and specific conserved residues of Sbp1 in translation repression activity. Method: Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions was created in Sbp1 expressed under a galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon over-expression. The ability of Sbp1 to affect over expression mediated growth defect of other decapping activators was tested using growth assay. Live cell imaging was done to study localization of Sbp1 and its RRM-deletion mutants to RNA granules upon glucose starvation. Results: Mutation of several aromatic residues in the RGG-motif and that of the phosphorylation sites in the RRM domain of Sbp1 did not affect the growth defect phenotype. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect the ability of Sbp1 to cause growth defect. Moreover, absence of Sbp1 did not affect the growth defect phenotypes observed upon overexpression of decapping activators Dhh1 and Pat1. Strikingly deletion of both the RRM domains (RRM1 and RRM2) and not the RNP motifs within them compromised the growth defect phenotype. Sbp1 mutant lacking both RRM1 and RRM2 was highly defective in localizing to RNA granules. Conclusion: This study identifies an important role of RRM domains independent of RNP motif in Sbp1 repression activity.
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Koukiali, Anastasia, Makrina Daniilidou, Ilias Mylonis, Thomas Giannakouros, and Eleni Nikolakaki. "SR Protein Kinase 1 Inhibition by TAF15." Cells 12, no. 1 (December 28, 2022): 126. http://dx.doi.org/10.3390/cells12010126.
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Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins.
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Poornima, Gopalakrishna, Ravishankar Mythili, Priyabrata Nag, Sabnam Parbin, Praveen Kumar Verma, Tanweer Hussain, and Purusharth I. Rajyaguru. "RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6." RNA Biology 16, no. 9 (June 12, 2019): 1215–27. http://dx.doi.org/10.1080/15476286.2019.1621623.
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Corbin-Lickfett, Kara A., Stuart K. Souki, Melanie J. Cocco, and Rozanne M. Sandri-Goldin. "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." Journal of Virology 84, no. 13 (April 21, 2010): 6367–76. http://dx.doi.org/10.1128/jvi.00509-10.
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ABSTRACT 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.
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Ruiz-Jarabo, Carmen M., Noemí Sevilla, Mercedes Dávila, Gema Gómez-Mariano, Eric Baranowski, and Esteban Domingo. "Antigenic properties and population stability of a foot-and-mouth disease virus with an altered Arg-Gly-Asp receptor-recognition motif." Journal of General Virology 80, no. 8 (August 1, 1999): 1899–909. http://dx.doi.org/10.1099/0022-1317-80-8-1899.
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The antigenic properties and genetic stability of a multiply passaged foot-and-mouth disease virus (FMDV) clone C-S8c1 with an Arg-Gly-Gly triplet (RGG) instead of the Arg-Gly-Asp (RGD) integrin-recognition motif at positions 141 to143 of capsid protein VP1 are described. Clear antigenic differences between FMDV RGG and clone C-S8c1 have been documented in ELISA, enzyme-linked immunoelectrotransfer (Western) blot and neutralization assays using site A-specific monoclonal antibodies and anti-FMDV polyclonal antibodies from swine and guinea pigs. The results validate with a live virus the role of the RGD (in particular Asp-143) in recognition of (and neutralization by) antibodies, a role previously suggested by immunochemical and structural studies with synthetic peptides. The FMDV RGG was genetically stable in a large proportion of serial infections of BHK-21 cells. However, a revertant virus with RGD was generated in one out of six passage series. Interestingly, this revertant FMDV did not reach dominance but established an equilibrium with its parental FMDV RGG, accompanied by an increase of quasispecies complexity at the sequences around the RGG triplet. FMDV RGG exhibited a selective disadvantage relative to other RGD-containing clones isolated from the same parental FMDV population. The results suggest that large antigenic variations can be prompted by replacements at critical capsid sites, including those involved in receptor recognition. These critical replacements may yield viruses whose stability allows them to replicate efficiently and to expand the sequence repertoire of an antigenic site.
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Boulila, Younes, Stanislas Tomavo, and Mathieu Gissot. "A RGG motif protein is involved in Toxoplasma gondii stress-mediated response." Molecular and Biochemical Parasitology 196, no. 1 (August 2014): 1–8. http://dx.doi.org/10.1016/j.molbiopara.2014.07.009.
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Siomi, H., and G. Dreyfuss. "A nuclear localization domain in the hnRNP A1 protein." Journal of Cell Biology 129, no. 3 (May 1, 1995): 551–60. http://dx.doi.org/10.1083/jcb.129.3.551.
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The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta-galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.
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Yin, Ziwei, Maki Kobayashi, Wenjun Hu, Koichi Higashi, Nasim A. Begum, Ken Kurokawa, and Tasuku Honjo. "RNA-binding motifs of hnRNP K are critical for induction of antibody diversification by activation-induced cytidine deaminase." Proceedings of the National Academy of Sciences 117, no. 21 (May 8, 2020): 11624–35. http://dx.doi.org/10.1073/pnas.1921115117.
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Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, andcMyc/IgHtranslocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID–hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.
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Huang, Zhou-Li, Jing Dai, Wen-Hua Luo, Xiang-Gui Wang, Jia-Heng Tan, Shuo-Bin Chen, and Zhi-Shu Huang. "Identification of G-Quadruplex-Binding Protein from the Exploration of RGG Motif/G-Quadruplex Interactions." Journal of the American Chemical Society 140, no. 51 (December 5, 2018): 17945–55. http://dx.doi.org/10.1021/jacs.8b09329.
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Volná, Adriana, Martin Bartas, Jakub Nezval, Vladimír Špunda, Petr Pečinka, and Jiří Červeň. "Searching for G-Quadruplex-Binding Proteins in Plants: New Insight into Possible G-Quadruplex Regulation." BioTech 10, no. 4 (September 22, 2021): 20. http://dx.doi.org/10.3390/biotech10040020.
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G-quadruplexes are four-stranded nucleic acid structures occurring in the genomes of all living organisms and viruses. It is increasingly evident that these structures play important molecular roles; generally, by modulating gene expression and overall genome integrity. For a long period, G-quadruplexes have been studied specifically in the context of human promoters, telomeres, and associated diseases (cancers, neurological disorders). Several of the proteins for binding G-quadruplexes are known, providing promising targets for influencing G-quadruplex-related processes in organisms. Nonetheless, in plants, only a small number of G-quadruplex binding proteins have been described to date. Thus, we aimed to bioinformatically inspect the available protein sequences to find the best protein candidates with the potential to bind G-quadruplexes. Two similar glycine and arginine-rich G-quadruplex-binding motifs were described in humans. The first is the so-called “RGG motif”-RRGDGRRRGGGGRGQGGRGRGGGFKG, and the second (which has been recently described) is known as the “NIQI motif”-RGRGRGRGGGSGGSGGRGRG. Using this general knowledge, we searched for plant proteins containing the above mentioned motifs, using two independent approaches (BLASTp and FIMO scanning), and revealed many proteins containing the G4-binding motif(s). Our research also revealed the core proteins involved in G4 folding and resolving in green plants, algae, and the key plant model organism, Arabidopsis thaliana. The discovered protein candidates were annotated using STRINGdb and sorted by their molecular and physiological roles in simple schemes. Our results point to the significant role of G4-binding proteins in the regulation of gene expression in plants.
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Anderson, J. T., S. M. Wilson, K. V. Datar, and M. S. Swanson. "NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability." Molecular and Cellular Biology 13, no. 5 (May 1993): 2730–41. http://dx.doi.org/10.1128/mcb.13.5.2730-2741.1993.
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A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.
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Anderson, J. T., S. M. Wilson, K. V. Datar, and M. S. Swanson. "NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability." Molecular and Cellular Biology 13, no. 5 (May 1993): 2730–41. http://dx.doi.org/10.1128/mcb.13.5.2730.
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A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.
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Iacovides, Demetris C., Clodagh C. O'Shea, Juan Oses-Prieto, Alma Burlingame, and Frank McCormick. "Critical Role for Arginine Methylation in Adenovirus-Infected Cells." Journal of Virology 81, no. 23 (August 8, 2007): 13209–17. http://dx.doi.org/10.1128/jvi.01415-06.
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ABSTRACT During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.
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Liu, Q., and G. Dreyfuss. "In vivo and in vitro arginine methylation of RNA-binding proteins." Molecular and Cellular Biology 15, no. 5 (May 1995): 2800–2808. http://dx.doi.org/10.1128/mcb.15.5.2800.
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Heterogenous nuclear ribonucleoproteins (hnRNPs) bind pre-mRNAs and facilitate their processing into mRNAs. Many of the hnRNPs undergo extensive posttranslational modifications including methylation on arginine residues. hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. The role of this modification is not known. Here we identify the hnRNPs that are methylated in HeLa cells and demonstrate that most of the pre-mRNA-binding proteins receive this modification. Using recombinant human hnRNP A1 as a substrate, we have partially purified and characterized a protein-arginine N-methyltransferase specific for hnRNPs from HeLa cells. This methyltransferase can methylate the same subset of hnRNPs in vitro as are methylated in vivo. Furthermore, it can also methylate other RNA-binding proteins that contain the RGG motif RNA-binding domain. This activity is evolutionarily conserved from lower eukaryotes to mammals, suggesting that methylation has a significant role in the function of RNA-binding proteins.
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Solomon, Samuel, Yaoxian Xu, Bin Wang, Muriel D. David, Peter Schubert, Derek Kennedy, and John W. Schrader. "Distinct Structural Features ofCaprin-1 Mediate Its Interaction with G3BP-1 and Its Induction of Phosphorylation of Eukaryotic Translation InitiationFactor 2α, Entry to Cytoplasmic Stress Granules, and Selective Interaction with a Subset of mRNAs." Molecular and Cellular Biology 27, no. 6 (January 8, 2007): 2324–42. http://dx.doi.org/10.1128/mcb.02300-06.
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ABSTRACT Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G1-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2α (eIF-2α) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.
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Freischmidt, Axel, Anand Goswami, Katharina Limm, Vitaly L. Zimyanin, Maria Demestre, Hannes Glaß, Karlheinz Holzmann, et al. "A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis." Brain 144, no. 4 (April 1, 2021): 1214–29. http://dx.doi.org/10.1093/brain/awab018.
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Abstract Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.
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Yan, Qiuxia, Peng Zeng, Xiuqin Zhou, Xiaoying Zhao, Runqiang Chen, Jing Qiao, Ling Feng, Zhenjie Zhu, Guozhi Zhang, and Cairong Chen. "RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing." Oncogene 40, no. 15 (February 9, 2021): 2635–50. http://dx.doi.org/10.1038/s41388-021-01666-z.
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AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.
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Lorković, Z. J., R. G. Herrmann, and R. Oelmüller. "PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants." Molecular and Cellular Biology 17, no. 4 (April 1997): 2257–65. http://dx.doi.org/10.1128/mcb.17.4.2257.
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The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.
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Mears, W. E., and S. A. Rice. "The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation." Journal of virology 70, no. 11 (1996): 7445–53. http://dx.doi.org/10.1128/jvi.70.11.7445-7453.1996.
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Nakamoto, Meagan Y., Nickolaus C. Lammer, Robert T. Batey, and Deborah S. Wuttke. "hnRNPK recognition of the B motif of Xist and other biological RNAs." Nucleic Acids Research 48, no. 16 (August 19, 2020): 9320–35. http://dx.doi.org/10.1093/nar/gkaa677.
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Abstract Heterogeneous nuclear ribonuclear protein K (hnRNPK) is an abundant RNA-binding protein crucial for a wide variety of biological processes. While its binding preference for multi-cytosine-patch (C-patch) containing RNA is well documented, examination of binding to known cellular targets that contain C-patches reveals an unexpected breadth of binding affinities. Analysis of in-cell crosslinking data reinforces the notion that simple C-patch preference is not fully predictive of hnRNPK localization within transcripts. The individual RNA-binding domains of hnRNPK work together to interact with RNA tightly, with the KH3 domain being neither necessary nor sufficient for binding. Rather, the RG/RGG domain is implicated in providing essential contributions to RNA-binding, but not DNA-binding, affinity. hnRNPK is essential for X chromosome inactivation, where it interacts with Xist RNA specifically through the Xist B-repeat region. We use this interaction with an RNA motif derived from this B-repeat region to determine the RNA-structure dependence of C-patch recognition. While the location preferences of hnRNPK for C-patches are conformationally restricted within the hairpin, these structural constraints are relieved in the absence of RNA secondary structure. Together, these results illustrate how this multi-domain protein's ability to accommodate and yet discriminate between diverse cellular RNAs allows for its broad cellular functions.
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Zinszner, H., J. Sok, D. Immanuel, Y. Yin, and D. Ron. "TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling." Journal of Cell Science 110, no. 15 (August 1, 1997): 1741–50. http://dx.doi.org/10.1242/jcs.110.15.1741.
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TLS, the product of a gene commonly translocated in liposarcomas (TLS), is prototypical of a newly identified class of nuclear proteins that contain a C-terminal domain with a distinct RNA recognition motif (RRM) surrounded by Arg-Gly-Gly (RGG) repeats. Its unique N terminus serves as an essential transforming domain for a number of fusion oncoproteins in human sarcomas and leukemias. In this study we use an in vivo UV crosslinking procedure to probe the interactions of TLS with RNA. TLS is found to bind RNA in vivo and the association of TLS with RNA is rapidly diminished by treating cells with transcriptional inhibitors. This suggests that the species bound by TLS turns over rapidly. Surprisingly, the RRM was found to be dispensable for RNA binding by TLS in vivo, suggesting that at any one time most of the interactions between TLS and RNA in the cell are not sequence specific. Analysis of inter specific heterokaryons formed between human and mouse or Xenopus cells revealed that TLS engages in rapid nucleocytoplasmic shuttling, a finding confirmed by the ability of anti-TLS antibodies to trap TLS when injected into the cytoplasm of HeLa cells. Cellular fractionation experiments suggest that TLS binds to RNA in both the nucleus and cytoplasm and support the hypothesis that TLS functions as a heterogeneous ribonuclear protein (hnRNP)-like chaperone of RNA. These findings are discussed in the context of the role altered forms of TLS play in cellular transformation.
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Jenning, Madeleine, Bianka Marklein, Jimmy Ytterberg, Roman A. Zubarev, Vijay Joshua, Dirkjan van Schaardenburg, Lotte van de Stadt, et al. "Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection—a missing link for ACPA production." Annals of the Rheumatic Diseases 79, no. 9 (June 12, 2020): 1194–202. http://dx.doi.org/10.1136/annrheumdis-2019-216919.
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ObjectivesPorphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA.MethodsRecombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated.ResultsRACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57–71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018).ConclusionsRACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.
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Soliman, Tarik M., and Saul J. Silverstein. "Identification of an Export Control Sequence and a Requirement for the KH Domains in ICP27 from Herpes Simplex Virus Type 1." Journal of Virology 74, no. 16 (August 15, 2000): 7600–7609. http://dx.doi.org/10.1128/jvi.74.16.7600-7609.2000.
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ABSTRACT The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an RNA-binding protein that performs multiple functions required for the expression of HSV-1 genes during a productive infection. One essential function involves shuttling between the nucleus and the cytoplasm. Some of the domains identified in ICP27 include a leucine-rich nuclear export sequence (NES), a nuclear localization signal, three KH-like RNA-binding domains, and an RGG-box type RNA-binding motif. To study the contribution of two of the essential domains in ICP27 to HSV gene expression, we generated recombinant herpesviruses carrying deleterious mutations in the NES and KH domains of ICP27. To accomplish this, we fused the green fluorescent protein (GFP) to ICP27 and utilized fluorescence as a marker to isolate recombinant herpesviruses. Fusion of GFP to wild-type ICP27 did not disturb its localization or function or significantly reduce virus yield. Analysis of HSV gene expression in cells infected with a recombinant virus carrying a point mutation in the first KH-like RNA-binding domain revealed that nuclear export of ICP27 was not blocked, and the expression of only a subset of ICP27-dependent late genes was affected. These findings suggest that individual KH-like RNA-binding motifs in ICP27 may be involved in binding distinct RNAs. Analysis of recombinant viruses carrying a lethal mutation in the NES of ICP27 was not accomplished because this mutation results in a strong dominant-negative phenotype. Finally, we demonstrate that shuttling by ICP27 is regulated by an export control sequence adjacent to its NES that functions like the inhibitory sequence element found adjacent to the NES of NS1 from influenza virus.
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Rajyaguru, Purusharth, and Roy Parker. "RGG motif proteins: Modulators of mRNA functional states." Cell Cycle 11, no. 14 (January 15, 2012): 2594–99. http://dx.doi.org/10.4161/cc.20716.
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Chaussee, Michael S., Gail L. Sylva, Daniel E. Sturdevant, Laura M. Smoot, Morag R. Graham, Robert O. Watson, and James M. Musser. "Rgg Influences the Expression of Multiple Regulatory Loci To Coregulate Virulence Factor Expression in Streptococcus pyogenes." Infection and Immunity 70, no. 2 (February 2002): 762–70. http://dx.doi.org/10.1128/iai.70.2.762-770.2002.
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ABSTRACT The human pathogen Streptococcus pyogenes secretes many proteins to the cell wall and extracellular environment that contribute to virulence. Rgg regulates the expression of several exoproteins including a cysteine protease (SPE B), a nuclease (MF-1), a putative nuclease (MF-3), and autolysin. The functional heterogeneity of Rgg-regulated exoproteins and the lack of a conserved regulatory motif in the promoter regions of the genes suggested that Rgg interacts with additional regulatory networks to influence gene expression. DNA microarrays were used to test this hypothesis by comparing genomewide transcript profiles of S. pyogenes NZ131 and isogenic derivative NZ131 rgg during the exponential phase of growth. Transcripts of known and putative virulence-associated genes were more abundant in the rgg mutant, including emm, scpA, orfX, scl1, hasAB, slo, sagA, ska, speH, grab, mac, mf-1, and mf-3. Increased transcription of emm, scpA, and orfX in the rgg mutant was associated with increased production of the corresponding proteins. Differences in the expression of virulence-associated genes were associated with changes in the expression of several regulatory genes, including mga, sagA, csrRS, and fasBCA. The results show that Rgg influences the expression of multiple regulatory networks to coregulate virulence factor expression in S. pyogenes.
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Rajyaguru, Purusharth, Meipei She, and Roy Parker. "Scd6 Targets eIF4G to Repress Translation: RGG Motif Proteins as a Class of eIF4G-Binding Proteins." Molecular Cell 45, no. 2 (January 2012): 244–54. http://dx.doi.org/10.1016/j.molcel.2011.11.026.
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Dammer, Eric B., Claudia Fallini, Yair M. Gozal, Duc M. Duong, Wilfried Rossoll, Ping Xu, James J. Lah, et al. "Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination." PLoS ONE 7, no. 6 (June 21, 2012): e38658. http://dx.doi.org/10.1371/journal.pone.0038658.
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Roy, Raju, Gitartha Das, Ishwarya Achappa Kuttanda, Nupur Bhatter, and Purusharth I. Rajyaguru. "Low complexity RGG-motif sequence is required for Processing body (P-body) disassembly." Nature Communications 13, no. 1 (April 19, 2022). http://dx.doi.org/10.1038/s41467-022-29715-5.
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AbstractP-bodies are conserved mRNP complexes that are implicated in determining mRNA fate by affecting translation and mRNA decay. In this report, we identify RGG-motif containing translation repressor protein Sbp1 as a disassembly factor of P-bodies since disassembly of P-bodies is defective in Δsbp1. RGG-motif is necessary and sufficient to rescue the PB disassembly defect in Δsbp1. Binding studies using purified proteins revealed that Sbp1 physically interacts with Edc3 and Sbp1-Edc3 interaction competes with Edc3-Edc3 interaction. Purified Edc3 forms assemblies, promoted by the presence of RNA and NADH and the addition of purified Sbp1, but not the RGG-deletion mutant, leads to significantly decreased Edc3 assemblies. We further note that the aggregates of human EWSR1 protein, implicated in neurodegeneration, are more persistent in the absence of Sbp1 and overexpression of EWSR1 in Δsbp1 leads to a growth defect. Taken together, our observations suggest a role of Sbp1 in disassembly, which could apply to disease-relevant heterologous protein-aggregates.
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Tunnicliffe, Richard B., William K. Hu, Michele Y. Wu, Colin Levy, A. Paul Mould, Edward A. McKenzie, Rozanne M. Sandri-Goldin, and Alexander P. Golovanov. "Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27." mBio 10, no. 5 (October 22, 2019). http://dx.doi.org/10.1128/mbio.02551-19.
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ABSTRACT Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo. Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity. IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.
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Bonucci, A., M. G. Murrali, L. Banci, and R. Pierattelli. "A combined NMR and EPR investigation on the effect of the disordered RGG regions in the structure and the activity of the RRM domain of FUS." Scientific Reports 10, no. 1 (December 2020). http://dx.doi.org/10.1038/s41598-020-77899-x.
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AbstractStructural disorder represents a key feature in the mechanism of action of RNA-binding proteins (RBPs). Recent insights revealed that intrinsically disordered regions (IDRs) linking globular domains modulate their capability to interact with various sequences of RNA, but also regulate aggregation processes, stress-granules formation, and binding to other proteins. The FET protein family, which includes FUS (Fused in Sarcoma), EWG (Ewing Sarcoma) and TAF15 (TATA binding association factor 15) proteins, is a group of RBPs containing three different long IDRs characterized by the presence of RGG motifs. In this study, we present the characterization of a fragment of FUS comprising two RGG regions flanking the RNA Recognition Motif (RRM) alone and in the presence of a stem-loop RNA. From a combination of EPR and NMR spectroscopies, we established that the two RGG regions transiently interact with the RRM itself. These interactions may play a role in the recognition of stem-loop RNA, without a disorder-to-order transition but retaining high dynamics.
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de Vries, Tebbe, William Martelly, Sébastien Campagne, Kevin Sabath, Chris P. Sarnowski, Jason Wong, Alexander Leitner, Stefanie Jonas, Shalini Sharma, and Frédéric H. T. Allain. "Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly." Proceedings of the National Academy of Sciences 119, no. 6 (January 31, 2022). http://dx.doi.org/10.1073/pnas.2114092119.
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Significance Pre-messenger RNA (pre-mRNA) splicing is a key regulatory step in gene expression. The splicing reaction is mediated by the spliceosome, a dynamic complex comprising five small nuclear ribonucleoproteins (snRNPs), which assembles onto each intron in multiple steps. We present detailed structural analysis and supporting functional data of an important protein–RNA interaction between human U1 and U2 snRNP. Our structure shows that an intrinsically disordered arginine-glycine (RGG/RG)–rich motif of a U2 snRNP subunit forms an RNA-sequence–specific connection with U1 snRNP. This study broadens the functional scope of unstructured RGG/RG-rich motifs in RNA binding proteins and provides a molecular basis of early steps of spliceosome assembly, which may help develop innovative therapeutic strategies against diseases originating from splicing defects.
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Johansson, Josefin, Sarah Lidéus, Carina Frykholm, Cecilia Gunnarsson, Filip Mihalic, Sanna Gudmundsson, Sara Ekvall, et al. "Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions." European Journal of Human Genetics, June 5, 2023. http://dx.doi.org/10.1038/s41431-023-01392-y.
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AbstractRNA binding motif protein X‐linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene’s importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.
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Vishal, Sonali S., Denethi Wijegunawardana, Muthu Raj Salaikumaran, and Pallavi P. Gopal. "Sequence Determinants of TDP-43 Ribonucleoprotein Condensate Formation and Axonal Transport in Neurons." Frontiers in Cell and Developmental Biology 10 (May 12, 2022). http://dx.doi.org/10.3389/fcell.2022.876893.
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Mutations in TDP-43, a RNA-binding protein with multiple functions in RNA metabolism, cause amyotrophic lateral sclerosis (ALS), but it is uncertain how defects in RNA biology trigger motor neuron degeneration. TDP-43 is a major constituent of ribonucleoprotein (RNP) granules, phase separated biomolecular condensates that regulate RNA splicing, mRNA transport, and translation. ALS-associated TDP-43 mutations, most of which are found in the low complexity domain, promote aberrant liquid to solid phase transitions and impair the dynamic liquid-like properties and motility of RNP transport granules in neurons. Here, we perform a comparative analysis of ALS-linked mutations and TDP-43 variants in order to identify critical structural elements, aromatic and charged residues that are key determinants of TDP-43 RNP transport and condensate formation in neurons. We find that A315T and Q343R disease-linked mutations and substitutions of aromatic residues within the α-helical domain and LARKS, show the most severe defects in TDP-43 RNP granule transport and impair both anterograde and retrograde motility. F313L and F313-6L/Y substitutions of one or both phenylalanine residues in LARKS suggest the aromatic rings are important for TDP-43 RNP transport. Similarly, W334F/L substitutions of the tryptophan residue in the α-helical domain, impair TDP-43 RNP motility (W334L) or anterograde transport (W334F). We also show that R293A and R293K mutations, which disrupt the only RGG in the LCD, profoundly reduce long-range, directed transport and net velocity of TDP-43 RNP granules. In the disordered regions flanking the α-helical domain, we find that F283Y, F397Y or Y374F substitutions of conserved GF/G and SYS motifs, also impair anterograde and/or retrograde motility, possibly by altering hydrophobicity. Similarly, ALS-linked mutations in disordered regions distant from the α-helical domain also show anterograde transport deficits, consistent with previous findings, but these mutations are less severe than A315T and Q343R. Overall our findings demonstrate that the conserved α-helical domain, phenylalanine residues within LARKS and RGG motif are key determinants of TDP-43 RNP transport, suggesting they may mediate efficient recruitment of motors and adaptor proteins. These results offer a possible mechanism underlying ALS-linked TDP-43 defects in axonal transport and homeostasis.
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Wu, Shan, Boon Heng Dennis Teo, Seng Yin Kelly Wee, Junjie Chen, and Jinhua Lu. "The GAR/RGG motif defines a family of nuclear alarmins." Cell Death & Disease 12, no. 5 (May 2021). http://dx.doi.org/10.1038/s41419-021-03766-w.
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AbstractThe nucleus is the target of autoantibodies in many diseases, which suggests intrinsic nuclear adjuvants that confer its high autoimmunogenicity. Nucleolin (NCL) is one abundant nucleolar autoantigen in systemic lupus erythematosus (SLE) patients and, in lupus-prone mice, it elicits autoantibodies early. With purified NCL, we observed that it was a potent alarmin that activated monocytes, macrophages and dendritic cells and it was a ligand for TLR2 and TLR4. NCL released by necrotic cells also exhibited alarmin activity. The NCL alarmin activity resides in its glycine/arginine-rich (GAR/RGG) motif and can be displayed by synthetic GAR/RGG peptides. Two more GAR/RGG-containing nucleolar proteins, fibrillarin (FBRL) and GAR1, were also confirmed to be novel alarmins. Therefore, the GAR/RGG alarmin motif predicts a family of nucleolar alarmins. The apparent prevalence of nucleolar alarmins suggests their positive contribution to tissue homeostasis by inducing self-limiting tissue inflammation with autoimmunity only occurring when surveillance is broken down.
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Garg, Mani, Debadrita Roy, and Purusharth I. Rajyaguru. "Low complexity RGG-motif containing proteins Scd6 and Psp2 act as suppressors of clathrin heavy chain deficiency." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, July 2022, 119327. http://dx.doi.org/10.1016/j.bbamcr.2022.119327.
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Kedersha, Nancy, Marc D. Panas, Christopher A. Achorn, Shawn Lyons, Sarah Tisdale, Tyler Hickman, Marshall Thomas, et al. "G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits." Journal of Cell Biology 212, no. 7 (March 28, 2016). http://dx.doi.org/10.1083/jcb.201508028.
Full textAbstract:
Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by binding to Caprin1 or USP10.
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Thandapani, Palaniraja, Jingwen Song, Valentina Gandin, Yutian Cai, Samuel G. Rouleau, Jean-Michel Garant, Francois-Michel Boisvert, et al. "Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes." eLife 4 (August 12, 2015). http://dx.doi.org/10.7554/elife.06234.
Full textAbstract:
G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.