Academic literature on the topic 'RGG-motif Protein'

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.

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.

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.

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.

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.

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.

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: 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.

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.

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.