Arginine methylation of Sam68 and SLM proteins negatively regulates their poly(U) RNA binding activity

Sam68 (Src substrate associated during mitosis) and its homologues, SLM-1 and SLM-2 (Sam68-like mammalian proteins), are RNA binding proteins and contain the arg-gly (RG) repeats, in which arginine residues are methylated by the protein arginine methyltransferase 1 (PRMT1). However, it remains unclear whether the arginine methylation affects an RNA binding. Here, we report that methylation of Sam68 and SLM proteins markedly reduced their poly(U) binding ability in vitro. The RG repeats of Sam68 bound poly(U), but arginine methylation of the RG repeats abrogated its poly(U) binding ability in vitro. Overexpression of PRMT1 increased arginine methylation of Sam68 and SLM proteins in cells, which resulted in a decrease of their poly(U) binding ability. The results suggest that the RG repeats conserved in Sam68 and SLM proteins may function as an auxiliary RNA binding domain and arginine methylation may eliminate or reduce an RNA binding ability of the proteins.     

Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain

Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated.     

The arginine-1493 residue in QRRGRTGR1493G motif IV of the hepatitis C virus NS3 helicase domain is essential for NS3 protein methylation by the protein arginine methyltransferase 1

The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg(1493) in the QRR(1488)GRTGR(1493)G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg(1488) is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.     

Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.     

A novel function for Sam68: enhancement of HIV-1 RNA 3' end processing

Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3' splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3' end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3' end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3' end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.     

Regulation of Arginine Methylation in Endothelial Cells: Role in Premature Senescence and Apoptosis

With the recent characterization of enzymes responsible for protein arginine methylation and demonstration that catabolic products of arginine methylation, such as asymmetric dimethylarginine (ADMA), are among the most powerful mechanisms of atherogenesis, developing endothelial dysfunction and cardiovascular complications in a variety of pathologic processes, the need for functional characterization of the methylation-demethylation processes becomes ever more urgent. Therefore, the aims of the present study were to refine the feedback regulation of protein arginine methylation using one of the heavily methylated proteins, an RNA-binding protein Sam68, as a prototype, to elucidate the relations between Sam68 methylation and tyrosine phosphorylation and the role of methylation in RNA binding and subcellular distribution, as well as the cellular consequences of reduced protein methylation. Screening pro-atherogenic substances known to induce endothelial dysfunction showed that ADMA did not affect the level of arginine methylation of Sam68, whereas peroxynitrite was a strong inhibitor of methylation. Adavanced glycation-modified collagen I, which accumulats in diabetes and induces formation of peroxynitrite and premature endothelial cell senescence, also inhibited arginine methylation of Sam68. When the level of arginine methylation of Sam68 was pharmacologically reduced, this did not affect its RNA binding or degree of tyrosine phosphorylation, but resulted in the predominantly nuclear hypomethylation pattern. Furthermore, protein hypomethylation resulted in the increased rate of apoptosis and premature senescence. This data may offer an additional explanation for the pro-apoptotic and senescence-accelerating action of peroxynitrite, a potent inhibitor of protein methylation.     

An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization

The human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the nuclear export of viral mRNA containing the Rev response element (RRE). Although several host proteins co-operating with Rev in viral RNA export have been reported, little is known about the innate host defense factors that Rev overcomes to mediate the nuclear export of unspliced viral mRNAs. We report here that an anti-apoptotic protein, HS1-associated protein X-1 (Hax-1), a target of HIV-1 Vpr, interacts with Rev and inhibits its activity in RRE-mediated gene expression. Co-expression of Sam68 emancipates Rev activity from Hax-1-mediated inhibition. Hax-1 does not bind to RRE RNA by itself, but inhibits Rev from binding to RRE RNA in vitro. The impact of Hax-1 on Rev/RRE interactions in vitro correlates well with the reduced level of RRE-containing mRNA in vivo. Immunofluorescence studies further reveal that Hax-1 and Rev are cytoplasmic and nuclear proteins, respectively, when expressed independently. However, in Hax-1 co-expressing cells, Rev is translocated from the nucleus to the cytoplasm, where it is co-localized with Hax-1 in the cytoplasm. We propose that over-expression of Hax-1, possibly through binding to Rev, may interfere with the stability/export of RRE-containing mRNA and target the RNA for degradation.     

Critical role for arginine methylation in adenovirus-infected cells

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.     

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.     

Suppression of receptor interacting protein 140 repressive activity by protein arginine methylation

Receptor interacting protein 140 (RIP140), a ligand-dependent corepressor for nuclear receptors, can be modified by arginine methylation. Three methylated arginine residues, at Arg-240, Arg-650, and Arg-948, were identified by mass spectrometric analysis. Site-directed mutagenesis studies demonstrated the functionality of these arginine residues. The biological activity of RIP140 was suppressed by protein arginine methyltransferase 1 (PRMT1) due to RIP140 methylation, which reduced the recruitment of histone deacetylases to RIP140 and facilitated its nuclear export by enhancing interaction with exportin 1. A constitutive negative (Arg/Ala) mutant of RIP140 was resistant to the effect of PRMT1, and a constitutive positive (Arg/Phe) mutation mimicked the effect of arginine methylation. The biological activities of the wild type and the mutant proteins were examined in RIP140-null MEF cells. This study uncovered a novel means to inactivate, or suppress, RIP140, and demonstrated protein arginine methylation as a critical type of modification for corepressor.     

Arginine methylation of the cellular nucleic acid binding protein does not affect its subcellular localization but impedes RNA binding

Cellular nucleic acid binding protein (CNBP) contains seven zinc finger (ZF) repeats and an arginine and glycine (RG) rich sequence between the first and the second ZF. CNBP interacts with protein arginine methyltransferase PRMT1. Full-length but not RG-deleted or mutated CNBP can be methylated. Treatment with a methylation inhibitor AdOx reduced CNBP methylation, but did not affect the concentrated nuclear localization of CNBP. Nevertheless, arginine methylation of CNBP appeared to interfere with its RNA binding activity. Our findings show that arginine methylation of CNBP in the RG motif did not change the subcellular localization, but regulated its RNA binding activity.Structured summary of protein interactionsPRMT1 binds to CNBP by pull down (View interaction)PRMT1 methylates CNBP by enzymatic study (View interaction)CNBP physically interacts with PRMT1 by anti tag coimmunoprecipitation (View interaction)     

Recent advances in protein methylation: Enzymatic methylation of nucleic acid binding proteins

Summary Heterogeneous nuclear RNP protein A1, one of the major proteins in hnRNP particle (precursor for mRNA), is known to be post-translationally arginine-methylatedin vivo on residues 193, 205, 217 and 224 within the RGG box, the motif postulated to be an RNA binding domain. Possible effect of N^G-arginine methyl-modification in the interaction of protein A1 to nucleic acid was investigated. The recombinant hnRNP protein A1 wasin vitro methylated by the purified nuclear protein/histone-specific protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase) stoichiometrically and the relative binding affinity of the methylated and the unmethylated protein A1 to nucleic acid was compared: Differences in their binding properties to ssDNA-cellulose, pI values and trypsin sensitivities in the presence and absence of MS2-RNA all indicate that the binding property of hnRNP protein A1 to single-stranded nucleic acid has been significantly reduced subsequent to the methylation. These results suggest that posttranslational methyl group insertion to the arginine residue reduces protein-RNA interaction, perhaps due to interference of H-bonding between guanidino nitrogen arginine and phosphate RNA.Abbreviations hnRNP heterogeneous ribonucleoprotein particle - AdoMet S-adenosyl-L-methionine - AdoHcy S-adenosyl-L-homocysteine - MBP myelin basic protein - HMG high mobility group - ss single stranded