Arg-rich Domains Research Articles

Middle-Down Proteomics Reveals Dense Sites of Methylation and Phosphorylation in Arginine-Rich RNA-Binding Proteins.

Post-translational modifications (PTMs) within arginine (Arg)-rich RNA-binding proteins, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron-transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of human embryonic kidney (HEK)-293T cells. Notably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicates that phosphorylation and methylation do not often co-occur, suggesting that they may functionally oppose one another. Furthermore, we show that phosphorylation may modify interactions between Arg-rich proteins, as serine-arginine splicing factor 2 (SRSF2) has a stronger association with U1-70K and LUC7L3 upon dephosphorylation. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome and a possible unexplored regulator of RNA-binding protein interactions.

The Gly–Ala repeat modulates the interaction of Epstein–Barr virus nuclear antigen-1 with cellular chromatin

The Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA1) plays a pivotal role in EBV infection by anchoring the viral episome to cellular DNA, which regulates replication and partitioning in dividing cells. Here, we have used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques to study the interaction of EBNA1 with cellular chromatin in interphase and mitosis. This analysis revealed that while EBNA1 is highly mobile in both conditions, mobility is significantly reduced in mitosis when an immobile fraction is also detected. The N-terminal chromatin-targeting module of EBNA1 includes two Gly–Arg rich domains (GR1 and GR2) separated by a Gly–Ala repeat (GAr) of variable length. Using a set of deletion mutants and GFP-fusion reporters, we found that the GR domains cooperatively determine the mobility of EBNA1, whereas mobility is increased by the interposed GAr in a length-dependent manner. These findings highlight a previously unrecognized property of the interaction of EBNA1 with cellular chromatin that may fine-tune its function in the maintenance of viral latency.

Accurate Localization and Relative Quantification of Arginine Methylation Using Nanoflow Liquid Chromatography Coupled to Electron Transfer Dissociation and Orbitrap Mass Spectrometry

Protein arginine (Arg) methylation serves an important functional role in eucaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron-transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679-695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z = 2-4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.

The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA

Binding of coat protein (CP) to the 3′ nontranslated region (3′-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3′-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3′-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3′-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg 2+, lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3′-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3′-NTR level in Alfamo- and Ilarvirus genera.

Suppression of an intrinsic strand transfer activity of HIV-1 Tat protein by its second-exon sequences

The Tat protein of human immunodeficiency virus type 1 (HIV-1) has been shown to restrict premature reverse transcription at late stages of virus infection and to thus ensure the integrity of the viral RNA genome for packaging. To gain further insights into the roles of Tat in HIV-1 reverse transcription, we have assessed its effects on the first-strand transfer during the synthesis of minus-strand DNA through use of a reconstituted cell-free system. The results demonstrated that a form of Tat, containing only the first exon (Tat72), was able to enhance the first-strand transfer as efficiently as did the viral nucleocapsid protein. Coincidentally, this form of Tat was unable to inhibit the production of minus-strand strong-stop DNA. Further studies with various mutated forms of Tat showed that its Cys-rich region, rather than the core and Arg-rich domains, was essential for this strand transfer activity. Moreover, this activity of Tat is largely independent of the TAR RNA structure. Although full-length Tat protein (Tat86) was also able to promote strand transfer, this activity was limited by a strong overall inhibition of reverse transcription because of the presence of the second Tat exon. Other nucleic-acid-binding proteins (e.g., single-strand DNA-binding protein) were employed as negative controls and were unable to promote strand transfer in these assays. We propose that Tat possesses nucleic acid chaperone activity and can promote the first-strand transfer during HIV-1 reverse transcription; however, these activities are restricted by the second Tat exon, and the roles of these Tat activities in viral replication remain to be elucidated.

Heterologous basic domain substitutions in the HIV-1 Tat protein reveal an arginine-rich motif required for transactivation.

The Tat protein coded by HIV-1 is a unique eukaryotic transactivator. It activates gene expression from the viral LTR by its interaction with a nascent RNA element (TAR) located at the 5' end of all HIV-1 transcripts. Tat appears to bind to its target RNA structure in a highly sequence-specific manner. The TAR-binding activity of Tat has been localized in an Arg-rich basic domain located between residues 49 and 57 of the Tat protein. We have carried out domain substitution studies with heterologous basic domains which are also implicated in RNA binding. Here, we report that a 19 or a 12 amino acid region from the N-terminus of HTLV-I Rex can functionally substitute for the Tat basic domain. In contrast, the Arg-rich domains of the N gene products of bacteriophages lambda and 21 do not functionally substitute for the Tat basic domain. The positive and negative effects of various domain substitution mutants have facilitated identification of a consensus sequence (Arg/Lys-X-X-Arg-Arg-X-Arg-Arg) in the basic domain required for Tat activity. Conversion of the functionally inactive basic domain of the lambda N protein to the consensus motif restored the transactivation function of the Tat-N chimeric protein. Similarly, the Rex basic domain containing scrambled sequences unrelated or partially related to the consensus motif were either totally defective in transactivation or exhibited reduced activity. Our results further suggest that the activity of the core Arg motif may be enhanced by the presence of Gln or Asn within the basic domain.(ABSTRACT TRUNCATED AT 250 WORDS)