Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B′. L292 mouse fibroblasts were labeled in vivo with [³H]methionine. Sm D1, Sm D3, and Sm B/B′ were purified from either nuclear extracts, cytosolic extracts or a cytosolic 6S complex by immunoprecipitation of the Sm protein-containing complexes and then separation by electrophoresis on a polyacrylamide gel containing urea. The isolated Sm D1, Sm D3 or Sm B/B′ proteins were hydrolyzed to amino acids and the products were analyzed by high-resolution cation exchange chromatography. Sm D1, Sm D3, and Sm B/B′ isolated from nuclear fractions were all found to contain ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B′ were also found to contain asymmetric ω-N^G,N^G-dimethylarginine in these nuclear fractions. Analysis of Sm B/B′ from cytosolic fractions and Sm B/B′ and Sm D1 from cytosolic 6S complexes showed only the presence of ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B′ are asymmetrically dimethylated and that these modified proteins are located in the nucleus. In reactions in which Sm D1 or Sm D3 was methylated in vitro with a hemagglutinin-tagged PRMT5 purified from HeLa cells, we detected both symmetric ω-N^G,N^G′-dimethylarginine and asymmetric ω-N^G,N^G-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric ω-N^G,N^G′-dimethylarginine when a sodium phosphate buffer was used. These results suggest that the activity responsible for the formation of asymmetric dimethylated arginine residues in Sm proteins is either PRMT5 or a protein associated with it in the immunoprecipitated complex.     

High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma

The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. L-Arginine is converted to NO and L-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-mul aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74+/-19 (SD), 0.46+/-0.09, and 0.37+/-0.07 microM in healthy humans (n=85), respectively, and 44+/-14, 0.72+/-0.23, and 0.19+/-0.06 microM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166+/-50 and 85+/-22, respectively.     

TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic ‘reader’ Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.     

Determination of dimethylated arginines in human plasma by high-performance liquid chromatography

Using high-performance liquid chromatography (HPLC) with multigradient elution, (asymmetric-DMA, ADMA) and (symmetric-DMA, SDMA) can be separated from human plasma samples. The dimethylarginine compounds in plasma, after extraction with a cation-exchange column, are converted to fluorescent derivatives with o-phthaldialdehyde (OPA) in an alkaline medium and the derivatives are separated simultaneously within 50 min on a reversed-phase column (Ultracarb 3 ODS(20)). The recoveries of ADMA and SDMA are over 80% and the method permits quantitative determination of dimethylated arginines at concentrations as low as 0.1 μmol/l in human plasma.     

High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8A crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.     

Structure and function of eTudor domain containing TDRD proteins

Tudor domain-containing (TDRD) proteins, as a family of evolutionarily conserved proteins, have been studied extensively in recent years in terms of their biological and biochemical functions. A major function of the TDRD proteins is to recognize the N-terminal arginine-rich motifs of the P-element-induced wimpy testis (PIWI) proteins via their conserved extended Tudor (eTudor or eTud) domains, which is essential in piRNA biogenesis and germ cell development. In this review, we summarize recent progress in the study of the TDRD proteins, and discuss the molecular mechanisms for the different binding selectivity of these eTudor domains to PIWI proteins based on the available binding and structural data. Understanding the binding differences of these TDRDs to PIWI proteins will help us better understand their functional differences and aid us in developing the target-specific therapeutics, because overexpression or mutations of the human TDRD proteins have been demonstrated to associate with various diseases.     

Recognition of 3D flexible objects by GRBF

Recently Poggio and Edelman have shown that for each object there exists a smooth mapping from an arbitrary view to its standard view and that the mapping can be learned from a sparse data set. In this paper, we extend their scheme further to deal with 3D flexible objects. We show the mappings from an arbitrary view to the standard view, and its rotated view can be synthesized even for a flexible object by learning from examples. To classify 3D flexible objects, we propose two methods, which do not require any special knowledge on the target flexible objects. They are: (1) learning the characteristic function of the object and (2) learning the view-change transformation. We show their performance by computer simulations. Received: 1 March 1993/Accepted in revised form: 7 June 1993     

Inactivation of chloroperoxidase by arginine

Inactivation of chloroperoxidase (CPO) from Caldariomyces fumago by arginine was investigated. It was found that the red native CPO solution was turned into a stable green species with a concomitant shift of the Soret band from 398 to 425 nm in the presence of arginine. The green CPO lost almost all of its catalytic activity, and this inactivation was irreversible.Differential UV–vis spectrophotometry was used to examine the binding properties of arginine to CPO. The formation of CPO-arginine (1:1) complex was highly pH-dependent. Fluorescence investigation revealed the exposure degree of prosthetic group increased. Kinetic analysis indicated that CPO has both a high affinity and specificity to arginine.This inactivation may be caused mainly by the binding of guanidinium group in arginine to the acid–base catalytic group Glu183 in CPO. The change of surrounding environment around heme induced by the interaction of heme propionates with arginine and the occupying of the sixth axial ligand position of heme iron by hydroxyl are also reasons bringing on this inactivation.     

Recognition of RhoA by Clostridium botulinum C3 exoenzyme

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.     

An allosteric inhibitor of protein arginine methyltransferase 3

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in?vitro. Here, we?report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC(50) value of 2.5?μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.     

Recognition of various arginine transfer ribonucleic acids with arginyl-tRNA synthetase purified from human placenta

Arginyl-tRNA synthetase has been purified approximately 550 fold from crude extract of human placenta by the following purification steps: Ammonium sulfate fractionation, chromatographies of DEAE-cellulose and CM-Sephadex and Sephadex G-100 gel filtration. Final preparation of this enzyme has specific activity of 123 nmole of arginyl-tRNA formed per mg of protein and was free from other aminoacyl-tRNA synthetase activities. Recognition of various arginine tRNAs with this enzyme was studied using kinetic analysis of arginylation of arginine tRNA and also arginine tRNA dependent ATP-PPi exchange reaction. Affinity of this enzyme with arginine tRNA was determine from Vmas/Km values and it was in the order of rabbit, Chum salmon, B. subtilis, E. coli and yeast in both systems.     

Two-Site Recognition of Phosphatidylinositol 3-Phosphate by PROPPINs in Autophagy

Macroautophagy is essential to cell survival during starvation and proceeds by the growth of a double-membraned phagophore, which engulfs cytosol and other substrates. The synthesis and recognition of the lipid phosphatidylinositol 3-phosphate, PI(3)P, is essential for autophagy. The key autophagic PI(3)P sensors, which are conserved from yeast to humans, belong to the PROPPIN family. Here we report the crystal structure of the yeast PROPPIN Hsv2. The structure consists of a seven-bladed β-propeller and, unexpectedly, contains two pseudo-equivalent PI(3)P binding sites on blades 5 and 6. These two sites both contribute to membrane binding in?vitro and are collectively required for full autophagic function in yeast. These sites function in concert with membrane binding by a hydrophobic loop in blade 6, explaining the specificity of the PROPPINs for membrane-bound PI(3)P. These observations thus provide a structural and mechanistic framework for one of the conserved central molecular recognition events in autophagy.