Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function. Cofactor binding and substrate interactions

Many eukaryotic RNA-binding proteins are modified by methylation of arginine residues. The yeast Saccharomyces cerevisiae contains one major arginine methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth. However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function. We now demonstrate that the catalytically active form of Hmt1p is required for its activity in vivo. Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1. In addition these mutations affect nucleocytoplasmic transport of Npl3p. A cold-sensitive mutant of Hmt1p was generated and showed reduced methylation of Npl3p, but not of other substrates, at 14 degrees C. These results define new aspects of Hmt1 and reveal the importance of its activity in vivo.     

The structure and oligomerization of the yeast arginine methyltransferase, Hmt1

Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting. Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus. We now report the crystal structure of Hmt1 at 2.9 A resolution. Hmt1 forms a hexamer with approximate 32 symmetry. The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces. Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro. Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.     

Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae

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.     

Novel RING finger proteins, Air1p and Air2p, interact with Hmt1p and inhibit the arginine methylation of Npl3p

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in the mRNA processing and export and are post-translationally modified by methylation at arginine residues in their arginine-glycine-rich (RGG) domains. We screened the factors that can interact with the RGG domain of Npl3p only in the presence of Hmt1p with the two-hybrid system in Saccharomyces cerevisiae. An isolated clone, YIL079, encodes a novel RING finger protein that was not directly bound to Npl3p but associated with the N terminus of Hmt1p. Thus, we designated the gene product Air1p (arginine methyltransferase-interacting RING finger protein). Air1p inhibited the Hmt1p-mediated methylation of Npl3p in vitro. Overexpression of Air1p repressed the Hmt1p-dependent growth of cells. Since homology searches indicate that the YDL175 gene product has significant identity (45%) with Air1p, we designated the gene AIR2. Air2p also has a RING finger domain and was bound to Hmt1p. Although single disruption of either gene gave no effect on the cell growth, cells lacking Air1p and Air2p grew at an extremely slow rate with accumulated poly(A)(+) RNA in the nucleus. Thus, Air1p and Air2p may affect mRNA transport by regulating the arginine methylation state of heterogeneous nuclear ribonucleoproteins.     

Nuclear export of hnRNP Hrp1p and nuclear export of hnRNP Npl3p are linked and influenced by the methylation state of Npl3p

Eukaryotic mRNA processing and export are mediated by a series of complexes composed of heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated at arginine residues within their RGG domains. Although cellular arginine methylation is required for the efficient nuclear export of several hnRNPs, its role in this process is unknown. To address this question, we replaced the methylated RGG tripeptides of two hnRNPs, Npl3p and Hrp1p, with KGG. We found that these substitutions specifically abolish their methylation but have different effects on their nuclear export activity. Although the efficient export of Hrp1p requires cellular methyltransferase activity, the modification of Hrp1p itself is dispensable. In contrast, we found that Npl3 arginine methylation not only facilitates its own export but also is required for Hrp1p to efficiently exit the nucleus. Consistent with this observation, we found that Npl3p and Hrp1p exist in a ribonucleoprotein complex. We provide the first evidence that the arginine methylation of a particular protein directly affects its activity. Efficient export does not require methylation per se, but unmethylated arginine residues lead to retention of hnRNPs. Thus, arginine methylation serves to mask the Npl3p RGG domain for efficient ribonucleoprotein export.     

Protein arginine methylation in Candida albicans: role in nuclear transport

Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and omega-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Delta strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Delta/rmt2Delta strain, as well as biochemical evidence for additional cryptic PRMTs.     

Expression of proteins with dimethylarginines in Escherichia coli for protein-protein interaction studies

Protein arginine methylation often modulates protein-protein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in omega-N(G),N(G)-asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His(6)-tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on protein-protein interaction.     

Specific sequences within arginine–glycine-rich domains affect mRNA-binding protein function

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine–glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation.     

In vivo analysis of nucleolar proteins modified by the yeast arginine methyltransferase Hmt1/Rmt1p

In this report, we have investigated the impact of arginine methylation on the Gar1, Nop1, and Nsr1 nucleolar proteins in Saccharomyces cerevisiae. Although previous reports have established that protein arginine methylation is important for nucleocytoplasmic shuttling, they have focused on the examination of heterogeneous nuclear ribonucleoproteins (hnRNPs). We have extended this analysis to several nucleolar proteins that represent a distinct functional class of arginine-methylated proteins. We first developed an in vivo assay to identify proteins methylated by the Hmt1 arginine methyltransferase. This assay is based on the fact that the Hmt1 enzyme utilizes S-Adenosyl-L-methionine as the methyl donor for protein arginine methylation. Following SDS polyacrylamide electrophoresis, 11 distinct proteins were identified as substrates for the Hmt1 methyltransferase. Hmt1p overexpression did not increase the methylation level on these proteins, suggesting they are fully methylated under the conditions examined. Three of the radiolabeled proteins were confirmed to be Gar1p, Nop1p, and Nsr1p. To monitor the cellular localization of these proteins, functional GFP fusion proteins were generated and found to be localized to the nucleolus. This localization was independent of arginine methylation. Furthermore, all three proteins examined did not export to the cytoplasm. In contrast, arginine methylation is required for the export of the nuclear RNA-binding proteins Npl3p, Hrp1p, and Nab2p. The observation that three nucleolar proteins are modified by Hmt1p but are not exported from the nucleolus implies an alternate role for arginine methylation.     

Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide

The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.     

A conditional two‐hybrid (C2H) system for the detection of protein–protein interactions that are mediated by post‐translational modification

The original bacterial two‐hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two‐hybrid (C2H) system, in which bait and prey proteins can be co‐expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co‐expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S‐tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.     

Biochemical analysis of the arginine methylation of high molecular weight fibroblast growth factor-2

The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -22, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions.     

Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding,its ability to suppress stress granule formation,and viral replication

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5’-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein–mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.     

Scd6 targets eIF4G to repress translation: RGG motif proteins as a class of eIF4G-binding proteins

The formation of mRNPs controls the interaction of the translation and degradation machinery with individual mRNAs. The yeast Scd6 protein and its orthologs regulate translation and mRNA degradation in yeast, C.?elegans, D.?melanogaster, and humans by an unknown mechanism. We demonstrate that Scd6 represses translation by binding the eIF4G subunit of eIF4F in a manner dependent on its RGG domain, thereby forming an mRNP repressed for translation initiation. Strikingly, several other RGG domain-containing proteins in yeast copurify with eIF4E/G and we demonstrate that two such proteins, Npl3 and Sbp1, also directly bind eIF4G and repress translation in a manner dependent on their RGG motifs. These observations identify the mechanism of Scd6 function through its RGG motif and indicate that eIF4G plays an important role as a scaffolding protein for the recruitment of translation repressors.     

Protein arginine methylation of SERBP1 by protein arginine methyltransferase 1 affects cytoplasmic/nuclear distribution

Protein arginine methylation regulates a broad array of cellular processes. SERBP1 implicated in tumor progression through its putative involvement in the plaminogen activator protease cascade, is an RNA-binding protein containing an RG-rich domain and an RGG box domain that might be methylated by protein arginine N-methyltransferases (PRMTs). Asymmetric dimethylarginine (aDMA) was detected in SERBP1 and an indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) significantly reduced the methylation signals. Arginines in the middle RG and C-terminal RGG region of SERBP1 are methylated based on the analyses of different deletion constructs. The predominant type I protein arginine methyltransferase PRMT1 co-immunoprecipitated with SERBP1 and the level of bound PRMT1 decreased upon the addition of AdOx. Recombinant PRMT1 methylated SERBP1 and knockdown of PRMT1 significantly reduced the aDMA level of SERBP1, indicating that SERBP1 is specifically methylated by PRMT1. Immunofluorescent analyses of endogenous SERBP1 showed predominant cytoplasmic localization of SERBP1. Treatment of AdOx or PRMT1 siRNA increased the nuclear localization of SERBP1. Analyses of different deletions indicated that the middle RG region is important for the nuclear localization while both N- and C- terminus are required for nuclear export. Low methylation of the C-terminal RGG region also favors nuclear localization. In conclusion, the RG-rich and RGG box of SERBP1 is asymmetrically dimethylated by PRMT1 and the modification affects protein interaction and intracellular localization of the protein. These findings provide the basis for dissecting the roles of SERBP1.