RNase treatment of yeast and mammalian cell extracts affects in vitro substrate methylation by type I protein arginine N-methyltransferases.

Type I protein arginine N-methyltransferases catalyze the formation of omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine residues using S-adenosyl-l-methionine as the methyl donor. In vitro these enzymes can modify a number of soluble methyl-accepting substrates in yeast and mammalian cell extracts including several species that interact with RNA. We treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell extracts with RNase prior to in vitro methylation by recombinant protein N-arginine methyltransferases and found that the methylation of certain polypeptides is enhanced up to 12-fold whereas that of others is diminished. 2-D gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1, Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric points and apparent molecular weights. These results suggest that the methylation and RNA-binding of GAR domain-containing proteins in vivo may regulate protein-nucleic acid or protein-protein interactions.     

Role for Btg1 and Btg2 in growth arrest of WEHI-231 cells through arginine methylation following membrane immunoglobulin engagement

Engagement of membrane Ig (mIg) on WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells, results in growth arrest and subsequent apoptosis. Of the several dozen genes upregulated greater than two-fold by anti-IgM treatment through DNA microarray analysis, we focused on B cell translocation gene 1 (Btg1) and Btg2, member of Btg/Tob family of proteins. WEHI-231 cells were infected with the Btg1/EGFP or Btg2/EGFP retroviral vectors, and those expressing either Btg1 or Btg2 accumulated in G1 phase at significantly higher proportions than that seen for cells expressing control vector. Btg1 or Btg2 bound to protein arginine methyltransferase (PRMT) 1 via the box C region, an interaction required for anti-IgM-induced growth inhibition. The arginine methyltransferase inhibitor AdOx partially abrogated growth inhibition induced by Btg1, Btg2, or anti-IgM. The Btg1- or Btg2-induced growth inhibition was also abrogated in PRMT1-deficient cells via introduction of small interference RNA. In addition, we observed anti-IgM-induced arginine methylation of two proteins, a 28-kDa and a 36-kDa protein. Methylation, detected by a monoclonal antibody specific for asymmetric, but not symmetric methyl residues, was observed as early as 1 h-2 h after stimulation and was sustained for up to 24 h. The anti-IgM-induced p36 arginine methylation was abrogated in the PRMT1-deficient cells, suggesting that PRMT1 induces p36 methylation. Together, these results suggest that anti-IgM-induced growth inhibition is mediated via upregulation of Btg1 and Btg2, resulting in the activation of arginine methyltransferase activity and culminating in growth inhibition of WEHI-231 cells.     

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.     

RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity

Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins.     

Stm1p, a G4 quadruplex and purine motif triplex nucleic acid-binding protein, interacts with ribosomes and subtelomeric Y' DNA in Saccharomyces cerevisiae

The Saccharomyces cerevisiae protein Stm1 was originally identified as a G4 quadruplex and purine motif triplex nucleic acid-binding protein. However, more recent studies have suggested a role for Stm1p in processes ranging from antiapoptosis to telomere maintenance. To better understand the biological role of Stm1p and its potential for G(*)G multiplex binding, we used epitope-tagged protein and immunological methods to identify the subcellular localization and protein and nucleic acid partners of Stm1p in vivo. Indirect immunofluorescence microscopy indicated that Stm1p is primarily a cytoplasmic protein, although a small percentage is also present in the nucleus. Conventional immunoprecipitation found that Stm1p is associated with ribosomal proteins and rRNA. This association was verified by rate zonal separation through sucrose gradients, which showed that Stm1p binds exclusively to mature 80 S ribosomes and polysomes. Chromatin immunoprecipitation experiments found that Stm1p preferentially binds telomere-proximal Y' element DNA sequences. Taken together, our data suggest that Stm1p is primarily a ribosome-associated protein, but one that can also interact with DNA, especially subtelomeric sequences. We discuss the implications of our findings in relation to prior genetic, genomic, and proteomic studies that have identified STM1 and/or Stm1p as well as the possible biological role of Stm1p.     

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.     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.     

人细胞周期蛋白G2基因真核表达载体构建及其功能研究

构建人cyclin G2基因真核表达载体,进一步研究cyclin G2对体外培养细胞增殖的调节作用及可能的调节机制。以人口腔癌前上皮细胞系POE4总RNA的反转录产物为模板,应用RT-PCR方法克隆cyclin G2基因cDNA,成功构建真核表达载体pIRES -G2;应用脂质体介导的基因转染技术,以体外培养的肿瘤细胞系HeLa细胞和正常细胞系CV-1细胞作为受体细胞,进行转基因表达研究,发现cyclin G2高表达对体外培养细胞的增殖起明显抑制作用;应用p16INK4a、p21WAF1、p27KIP1三种周期蛋白依赖性激酶抑制因子的单克隆抗体对转基因的HeLa细胞进行免疫细胞化学研究,发现转染pIRES-G2的实验组细胞中,p21 WAF1蛋白染色阳性细胞数明显多于转染空载体的对照组,平均光密度值高于对照组,两组间均有显著性差异（p<0.01）,提示cyclin G2抑制细胞增殖作用可能是通过诱导p21WAF1的表达而实现。     

Readers of histone methylarginine marks

Arginine methylation is a common posttranslational modification (PTM) that alters roughly 0.5% of all arginine residues in the cells. There are three types of arginine methylation: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). These three PTMs are enriched on RNA-binding proteins and on histones, and also impact signal transduction cascades. To date, over thirty arginine methylation sites have been cataloged on the different core histones. These modifications alter protein structure, impact interactions with DNA, and also generate docking sites for effector molecules. The primary “readers” of methylarginine marks are Tudor domain-containing proteins. The complete family of thirty-six Tudor domain-containing proteins has yet to be fully characterized, but at least ten bind methyllysine motifs and eight bind methylarginine motifs. In this review, we will highlight the biological roles of the Tudor domains that interact with arginine methylated motifs, and also address other types of interactions that are regulated by these particular PTMs. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

UPF1甲基化和miR-744-5p/CCND1在甲状腺乳头状癌中的作用机制研究

摘要 目的：探讨UPF1甲基化和miR-744-5p/CCND1在甲状腺乳头状癌中的作用机制研究。方法：将人甲状腺乳头状癌细胞株TCP-1和正常甲状腺上皮细胞Nthy-ori-3分别用去甲基化试剂5-Aza-CdR进行干预,分别在干预前后采用甲基化特异性PCR技术检测UPF1基因甲基化变化,采用Western-Blotting 检测干预UPF1、DNMT1、miR-744-5p、CCND1蛋白相对表达,采用transwell细胞侵袭实验检测细胞侵袭情况。结果：PCR扩增显示,UPF1基因在Nthy-ori-3组仅出现非甲基化引物扩增条带（U条带）,在TCP-1组仅出现甲基化引物扩增条带（M条带）。经5-Aza-Cdr作用后,UPF1基因甲基化扩增条带减少,甲基化表达降低。各组UPF1、DNMT1、miR-744-5p、CCND1蛋白相对表达差异具有统计学意义（P＜0.05）。与Nthy-ori-3组比较,TCP-1组DNMT1、UPF1蛋白相对表达明显提高,miR-744-5p、CCND1蛋白相对表达明显降低（P＜0.05）;与TCP-1组比较,TCP-1干预组DNMT1、UPF1蛋白相对表达明显降低,miR-744-5p、CCND1蛋白相对表达明显提高（P＜0.05）。与TCP-1组比较,TCP-1干预组细胞侵袭、迁移数量明显减少（P＜0.05）。结论：UPF1甲基化存在于甲状腺乳头状癌中,UPF1基因甲基化的表达缺失可能抑制miR-744-5p/CCND1轴,在甲状腺乳头状癌发生发展中发挥关键作用。     

Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p17 of B- and C-subtypes

A simple and efficient method for expression in Escherichia coli and purification of matrix protein, p17, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described. DNA sequences encoding p17 of B- and C-subtype were cloned from respective gag sequences. The gag sequences were obtained by PCR amplification using DNA extracted from peripheral blood lymphocytes of an HIV-1 infected patient from India. A T7-promoter-based expression system was optimized for expression of p17 in soluble form. p17 (B- and C-subtype) was purified to near homogeneity using conventional chromatographic techniques. Purification of p17 (C-subtype) is described for the first time with yield of 7.7 mg from a 1-liter culture. The yield of p17 (B-subtype) is 14.7 mg from a 1-liter culture, which is severalfold better than that reported earlier. N-terminal sequencing and CD spectra of the purified proteins, p17B and p17C, show that the proteins are properly processed and well-folded. The immunoreactivity of both types of p17 to sera from HIV-infected individuals is comparable.     

Promoter methylation of O(6)-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.     

Posttranslational arginine methylation of lamin A/C during myoblast fusion

Protein arginine methylation is a major posttranslational modification that regulates various cellular functions, such as RNA processing and DNA repair. A recent report showed the involvement of protein arginine methyltransferase (PRMT) 4 in chromatin remodeling and gene expression during muscle differentiation in C2C12 cells. Because the fusion of myoblasts is a unique phenomenon observed in skeletal muscle differentiation, the present study focused on the expression and activities of PRMTs during myoblast fusion in primary rat skeletal muscle. N(G), N(G)-asymmetric dimethylarginines (aDMA) and N(G), N'(G)-symmetric dimethylarginines (sDMA) were both found consistently throughout myoblast fusion. However, PRMT1 exhibited the highest activity during myoblast fusion and maintained the elevated activity thereafter, whereas PRMT5 reached its highest activity only after myoblast fusion. To identify the proteins modified by such PRMTs, we conducted 2-dimensional electrophoresis (2-DE) of total proteins before and after myoblast fusion, and protein spots on the 2-DE gel immunoreactive for aDMA and sDMA were identified by mass spectrometric analysis. Among the proteins identified, lamin C2 was in particular observed to be dimethylated. Arginine methylation of lamin may therefore be important for muscle development and maintenance.     

细菌双杂交系统的优化及其应用

细菌双杂交系统是一种用于检测体内蛋白质互作的方法，该方法互补腺苷酸环化酶功能，通过检测细胞表达的β-半乳糖苷酶LacZ的活性，分析蛋白质互作能力。但在应用过程中，发现存在操作繁琐、灵敏度低、难实现高通量操作等缺陷。本研究目的是对原有细菌双杂交进行优化，建立一种操作方便、可批量操作、具有较高灵敏度和能够实现实时监测的细菌双杂交系统，本研究成功构建由lacZ启动子控制的luxCDABE的报告质粒pBBR-lacZ-luxCDABE，引入原有的检测系统，新的判读标准并不影响原有的判读标准，本文通过快速灵敏地冷光观察菌落或测定菌液冷光值即可判读结果，优化后的双杂交系统，阳性对照组冷光值为阴性对照组的几十（霍乱弧菌为宿主）至几百倍（大肠杆菌为宿主），阴阳性结果判读差异明显，不仅节约实验成本，提高工作效率，又实现了连续监测。该系统不仅以大肠杆菌为宿主获得成功应用，宿主范围也扩展到霍乱弧菌。该系统通过质粒将lacZ启动子的拷贝数提高，减弱了非腺苷三磷酸（cAMP）因素引起的lacZ启动子活性的干扰，无需繁琐耗时的实验操作，为大批量的蛋白质互作分析及互作蛋白质的筛选提供了有利工具。     

Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src

Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.     

Peptide backbone conformation affects the substrate preference of protein arginine methyltransferase I

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.