Gotowa bibliografia na temat „RGG-motif Proteins”

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.

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.

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.

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.

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.

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.

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.

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.

Messenger RNA translation is one of the most energy expensive steps in the process of gene expression and therefore translation regulation is critical for maintaining cellular homeostasis. In contrast to transcriptional regulation, rapid cellular changes in protein levels and thus physiological homeostasis can be achieved by regulation of mRNAs at translation level. At every stage of its life cycle, mRNA associates with RNA-binding proteins. The dynamic remodeling of mRNPs plays a major role in translation control and deciding mRNA fate. In yeast, a conserved translation initiation factor eIF4G, which promotes translation activation, was discovered to act as a nexus for binding of negative regulators of translation. A subset of RGG-motif containing proteins bind to eIF4G and repress mRNA translation in yeast. RGG-motifs are low-complexity sequences that contain repeats of arginine and glycine residues. Translation repressor protein Scd6, targets eIF4G1 but does not alter the eIF4F complex. It represses mRNA translation by inhibiting the 48S complex formation on mRNA. Although the mechanism of repression by Scd6 protein has been reported, the regulatory events that may control the repression activity of RGG-motif proteins was not known. In our first study, we uncovered the role of arginine methylation in modulating the translation repression activity of a conserved RGG-motif protein, Scd6. We found that Hmt1, a conserved arginine methyltransferase, binds Scd6 and methylates it at the RGG-motif. Scd6-eIF4G1 interaction was compromised upon deletion of Hmt1 or mutating arginines in the RGG-motif. Based on these results, we concluded that arginine methylation is a key event that augments the mRNA translation repression activity of Scd6 by promoting its interaction with eIF4G1. This study led us to ask an intriguing question about mechanism(s) that could keep the repression activity of Scd6 in check when the target mRNAs need to be translated. Scd6 C-terminal is rich in RG/RGG and Q/N repeats. Such repeat sequences render a low complexity to Scd6 C-terminus. In our second study, we have uncovered the role of RGG-motif in translation control through its self-association that competes with eIF4G binding. We demonstrated that Scd6 binds itself in an RNA-independent manner both in vivo and in vitro. We further elucidated that Scd6 self-interaction competes with eIF4G1 binding and arginine methylation of Scd6 RGG-motif by Hmt1 negatively affects self-association. For our third study in which we elucidate the role of RGG-motif containing mRNA export factor Gbp2 in translation control. Our study found that Gbp2 directly associates with eIF4G1 via its RGG-motif and RGG-motif of Gbp2 is important for its polysome association. We found that purified Gbp2 directly represses mRNA translation of a luciferase reporter mRNA, in vitro. We demonstrated the ability of Gbp2 to repress mRNA translation in vivo using a GFP-reporter mRNA tethering assay. Put together, this study hints at the role of an RGG-motif export factor in translation control.

Control of gene expression in eukaryotes is regulated at various steps such as transcription, translation and protein degradation. Translation repression of mRNA regulates protein levels and maintains cell homeostasis. Translation control allows for spatiotemporal regulation of gene expression which is required for development and differentiation in organisms. Deregulation of translation can result in disease conditions like cancer and neurodegenerative diseases. In yeast Saccharomyces cerevisiae, the RGG-motif protein Scd6 (Suppressor of Clathrin Deficiency 6) represses translation by binding eIF4G1 via its RGG domain and prevents formation of 48S pre-initiation complex. Scd6 consists of N-terminal Lsm domain, central FDF domain and C-terminal RGG domain. In this study, we assessed the contribution of other domains of Scd6 in its translation repression ability. Overexpression of Scd6 causes growth defect as a result of global translation repression. We observed that overexpression of Lsm domain deletion mutant could partially rescue the growth defect phenotype suggesting that Lsm domain might be contributing in Scd6 mediated translation repression. Deletion of FDF domain did not result in any significant change in the growth defect phenotype of Scd6 overexpression. Interestingly, both Lsm and RGG domains are necessary but insufficient to repress translation on their own. Lsm domains are conserved RNA binding domains. By mutating the putative RNA binding motif within the Lsm domain we observed a rescue from the growth defect phenotype of Scd6. Also, our preliminary results indicate that the RNA binding motif mutant of Lsm domain is defective in binding poly(U) RNA. We analyzed the translation repression ability of Lsm domain mutants by observing RNA granule formation under stress and non-stress conditions. We observe that the mutants are defective in localizing to granules. In addition, the mutant containing only Lsm domain localizes to nucleus like structure in non-stress condition and forms fewer RNA granules in the cytoplasm upon stress. Since Scd6 binds eIF4G1 to repress translation we analyzed the ability of Lsm domain lacking Scd6 mutant to interact with eIF4G1 in vivo. Our preliminary observations suggest that Scd6 mutant lacking Lsm domain is deficient in binding eIF4G1 in vivo. Considering all the observations from our studies, we propose a model in which Lsm domain of Scd6 helps in recognition of the mRNA target of Scd6 which is followed by eIF4G1-RGG domain interaction leading to translation repression.

RNA granules are conserved membraneless mRNP complexes that play an important role in determining mRNA fate by affecting translation repression and mRNA decay. Processing bodies (P-bodies) harbor enzymes responsible for mRNA decay and proteins involved in modulating translation. Although many proteins have been identified to play a role in P-body assembly, a bonafide disassembly factor remains unknown. We observed that Sbp1 with the help of its RGG-motif promotes P-body disassembly in S. cerevisiae. This study provides an example of the role of low complexity sequence in RNA granule disassembly. We have further explored the role of human RGG-motif containing proteins in regulation of translation. Here we studied LSM14A (human homolog of Scd6) in maintaining RNA granule dynamics and regulation of mRNA. We observe that LSM14A binds to human eIF4G and plays an important role in regulating the translation of certain mRNAs in response to genotoxic stress. Overall using a combination of yeast and human cell culture system, this study provides critical insight into the role of conserved low complexity sequences.

Cellular events that rely on translation regulation has been well established, however the molecular details of factors involved in bringing translation control remains inadequately explored. RNA binding proteins form an integral part of transcriptional and post-transcriptional gene regulation pathway. However, the principles that govern the activity of an RNA binding protein is poorly explored. In this thesis, a systematic investigation has been done to delineate the contribution of individual RRM domains and arginine methylation in the RGG motif of an RNA binding protein, Sbp1 towards its function. Chapter 3 demonstrated that role of arginine methylation of Sbp1 RGG motif towards its translation repression and decapping enhancing activity. Pull-down assays indicated that Sbp1 interaction with eIF4G1 decreases when the methylating enzyme, Hmt1 is absent or the Sbp1 RGG motif is deleted. We also learned that Sbp1 mono-methylation increases upon glucose starvation stress, which is known to cause global translation repression in yeast. Moreover, arginine methylation of Sbp1 was found to be crucial for driving decapping activators such as Dhh1 and Scd6 to RNA granules. Together, our results have established functional relevance of arginine methylation towards translation repression and decapping enhancing ability of RNA binding protein, Sbp1. Chapter 4 investigated the role of RRM domains of Sbp1 towards causing over-expression mediated growth defect and localizing to RNA granules. RRM domains are the most abundant RNA binding domain that harbor 6-8 amino acid consensus sequence involved in RNA binding. Our results have demonstrated that upon deleting both the RRM domains and not the RNP sequence, SBP1 over-expression mediated growth defect can be rescued. Moreover, ∆RRM 1+2 mutant of Sbp1 could not localize to RNA granules upon glucose starvation than wild-type. These observations suggest that Sbp1 RRM domains function via sequences outside the RNP motif, which is yet to be discovered. Chapter 5 describes novel genetic interaction of SBP1 with genes involved in Non-sense mediated mRNA decay (NMD) pathway. Over-expression mediated growth defect by SBP1 is augmented upon individual deletion of UPF1, UPF2 and UPF3. However, the augmented growth phenotype was not due to an increase in the protein level of Sbp1. Our study has established a link between translation repression and mRNA decay. To summarize, our study has identified: i) importance of arginine methylation of Sbp1 in regulating its function, ii) contribution of RRM domains of Sbp1 in causing over-expression mediated growth defect and localize to RNA granules upon stress and iii) genetic modulators of Sbp1 function. These studies have been done in budding yeast. Although, yeast does not display tissue level specificity observed in complex organisms, the molecular pathways are largely conserved. Principles that govern mRNA fate in yeast can form the basis for hypothesizing how certain factors might function in humans.

Shadoo (Sho) is a member of the prion protein (PrP) family, found mainly in the brain. PrP is well-known as the central agent involved in the prion diseases - a form of fatal neurodegenerative disease. Despite decades of intense research, the natural functions of PrP have not been clearly elucidated. The reasons for this are complex, but it is likely that there is a level of redundancy in the functions performed by PrP. In particular, it has been suggested that Sho may have overlapping functions with PrP and that both proteins can compensate for one another. Therefore, investigating the natural function/s of Sho may provide some insight into the roles that the non-pathogenic form of PrP may play in vivo. Although the natural function of Sho is currently unknown, recent studies point to it having a role in neural tube development and neuroprotection. Comparative genomics is a powerful technique for providing insight into functionally important protein domains. Comparing Sho protein sequences over the course of evolution highlighted a strongly conserved sequence at the beginning of the N-terminus. Literature-based data mining led me to hypothesize that it may constitute an RGG box, a known RNA binding motif. This hypothesis provides an interesting link to PrP, which can bind RNA, although an RNA binding function has not yet been identified. However, the RNA binding region of PrP, also located at the beginning of the N-terminal region, does not have the characteristics of an RGG box. This thesis reports the results of a series of studies designed to test the hypothesis that Sho has an RGG box and establish the plausibility that Sho may play a functional role as an RNA-binding protein. The RGG box of Sho has strong sequence similarity to the RGG box of the Fragile X Mental Retardation Protein (FMRP), which is known to bind a range of mRNAs and play an important role in neural plasticity. Comparison of the RGG boxes of Sho and FMRP reveals that, like many RNA binding domains, the RGG boxes of Sho and FMRP lie within disordered protein domains. This work examines the nature of these flexible protein domains using molecular dynamics (MD) simulations. The RGG box of FMRP has an affinity for G-quadruplex RNA, which is also the form of RNA that binds most strongly to PrP. Here, MD simulations and biophysical experiments are used to investigate whether Sho, too, binds G-quadruplex RNA. Binding of 5 different RNA transcripts to the RGG box regions of Sho and FMRP and the N-terminus RNA binding domain of PrP was explored through circular dichroism, fluorescence and surface plasmon resonance experiments. The results of both MD simulations and biophysical experiments show that a peptide derived from the RGG box region of Sho is capable of binding G-quadruplex RNA with physiologically relevant affinity. The Sho RGG box peptide binds to certain RNA transcripts with similar affinity to peptides comprising the FMRP RGG box and the RNA-binding region of PrP. However, some differences in RNA-binding affinities across all three peptides also indicate their ability to discriminate between different RNA targets. Overall, the findings reported here suggest that Sho is likely to function, in some capacity, as an RNA-binding protein. As Sho and PrP are capable of binding G-quadruplex RNA with similar affinities, it is possible that they share an RNA binding function. Given the primary location of Sho and PrP on the outer cell membrane it seems most plausible that they bind extracellular RNA in a signaling context. This finding is particularly interesting as recent research indicates that RNA may be a cofactor in the conversion of PrP to its disease-producing isoform. There is also growing interest in the role of extracellular RNA as a target for cell surface receptors. Future studies to identify likely RNA binding partners for Sho provide a promising avenue for elucidating the natural function of this protein.