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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1591 (2002) 1–10 www.bba-direct.com Identification of methylated proteins by protein arginine N-methyltransferase 1, PRMT1, with a new expression cloning strategy Kazuhiro Wada, Koichi Inoue, Masatoshi Hagiwara* Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan Received 13 December 2001; received in revised form 11 March 2002; accepted 4 April 2002 Abstract Methylation at arginines has recently come to attention as a posttranslational modification of proteins, which is implicated in processes from signaling, transcriptional activation, to mRNA processing. Here we report that several proteins extracted from HeLa cells were methylated by PRMT1 (protein arginine N-methyltransferease 1) even on a nitrocellulose membrane, while proteins from Escherichia coli are not methylated with this protein. Screening PRMT1 substrates from a Egt11-HeLa cDNA library, we found that more than half of the 48 cDNA clones obtained encode putative RNA-binding proteins that have RGG (arginine–glycine–glycine) motifs, such as hnRNP R (heterogeneous nuclear ribonucleoprotein R) and hnRNP K. We cloned two novel arginine methylation substrates, ZF5 (zinc finger 5) and p137GPI (GPI-anchor protein p137), which do not possess typical RGG motifs. We also cloned a novel protein that has RGG motifs, but does not have any other RNA-binding motifs. We tentatively termed this clone SAMT1 (substrate of arginine methyl transferase 1). A63-VLD-65 to AAA mutation of PRMT1 suppressed the methylation of recombinant SAMT1 and other RGG proteins in the HeLa extracts. This systematic screening of substrate proteins with the solid phase methylation reaction will contribute to identify new roles of PRMT family. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Arginine methylation; PRMT1; RGG motif; RNA-binding protein; Solid phase screening 1. Introduction larginine and asymmetric NG,NG-dimethylarginine residues, while type II enzymes catalyze the formation of NG-mono- Methylation of arginine residues by covalent modifica- methylarginine and symmetric NG,NVG-dimethylarginine. tions occurs in eukaryotic proteins during or shortly follow- The asymmetric dimethylarginine appears to be the more ing mRNA to protein translation. Evidence for posttransla- common form while symmetric dimethylarginine has so far tional methylation of arginine residues was first provided by been identified only in a few proteins, including myelin the presence of radioactive species chromatographing at basic protein (MBP) [5], and spliceosomal Sm proteins D1 positions near that of arginine in acid hydrolysates of and D3 (SmD1 and SmD3) [6]. Reported type I enzymes are isolated calf thymus nuclei proteins incubated with [meth- the yeast Hmt1p/Rmt1p [7] and the mammalian PRMT1 [8], 14 yl C]-S-adenosyl-L-methionine (SAM) [1]. The products of PRMT3 [9], and CARM1/PRMT4 [10]. PRMT1 and yeast the methylation reaction were determined to be NG-mono- Hmt1p/Rmt1p prefer to methylate arginine residues in methylarginine and NG,NG-dimethylarginine [2]. Protein glycine rich regions, which are found in several RNPs, arginine N-methyltransferases (PRMTs) catalyze the fibrillarin, and nucleolin [11]. However, CARM1/PRMT4 sequential transfer of methyl groups from SAM to the has little or no activity with substrates preferred by these guanidino nitrogens of arginine residues within proteins enzymes, and the best substrate reported for CARM1/ [3,4]. It now appears that there are at least two distinct PRMT4 is histone H3 [10]. JBP1/PRMT5 can methylate classes of protein arginine methyltransferases according to histone H2A and H4 and myelin basic protein (MBP) [11]. their substrate specificity and reaction products [4]. The Recently, a potential candidate for a type II enzyme, JBP1/ type I enzymes catalyze the formation of NG-monomethy- PRMT5, has been discovered in mammalian cells with an apparent homologue in yeast, Hsl7 [12]. Yet, its sequence is well conserved with other family members, the catalytic * Corresponding author. Tel.: +81-3-5803-5836; fax: +81-3-5803-5853. activity of PRMT2 has not yet been demonstrated [13]. The E-mail address: [email protected] (M. Hagiwara). conservation among protein arginine N-methyltransferases 0167-4889/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S0167-4889(02)00202-1 2 K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 may underscore the potential biological importance of this fonic acid [MOPS] (pH 7.2), 300 mM, NaCl, 2 mM ethyl- posttranslational modification. In addition to these, the type enediaminetetraacetic acid [EDTA], 1 mM phenylmethyl- III enzyme was also recently discovered in yeast, and this sulfonyl fluoride [PMSF], 0.1 Ag/ml leupeptin and 0.4 AM S- 3 3 catalyzes the monomethylation of the internal y-guanidino adenosyl-L-[methyl- H] methionine [ H-SAM] (specific nitrogen atom of arginine residues [14]. activity of 1.3 Ci/mmol; Amersham Biosciences, NJ) with PRMT1 knockout mouse has already been established, 0.02–0.04 Ag/Al recombinant PRMT1 or bovine serum and the phenotype of the homozygous null mutant is albumin [BSA] (as a negative control) and incubated at 30 embryonic lethal [15], suggesting that PRMT1 is required jC for 45 min. The methylation reaction was terminated by for very early stages of mouse development. Though recent adding 20 Al sodium dodecyl sulfate [SDS] sample buffer and evidence has implicated arginine-specific protein methyl- boiled for 5 min. Each 20 Al reaction solution was then loaded transferases in nuclear export of some hnRNP proteins and onto a 15% SDS-polyacrylamide gel, and separated by in several signaling pathways (reviewed in Ref. [4]), spe- electrophoresis. Gels were stained with Coomassie blue R- cific roles of protein methylation and the relevant protein 250 to visualize protein bands and then fluorographed, dried substrates have not been identified. Because each methyl- and exposed to Kodak film at À 80 jC for 2 days. transferase recognizes a different set of protein substrates, systematic analysis of their substrate specificity is now 2.2. Solid phase methylation assay required for differentiating their function. To this end, we report here a new expression cloning strategy to identify Cell extracts were size-separated on SDS-polyacrylamide substrate proteins of PRMT1, which is potentially applica- gel electrophoresis [SDS-PAGE] and transferred to a nitro- ble to isolate substrates specific for each member of the cellulose membrane. The membrane was then incubated in a PRMT family. reaction mix containing 50 mM Na-MOPS (pH 7.2), 300 mM NaCl, 2 mM EDTA, 1 mM PMSF, 0.1 Ag/ml leupeptin, and 0.4 AM 3H-SAM with 1 Ag of recombinant PRMT1 at 2. Materials and methods 30 jC for 1 h, washed with PBS [phosphate-buffered saline] and TPBS [Tris–phosphate-buffered saline], fluorographed, 2.1. In vitro methylation reaction dried, and exposed to Kodak film at À 80 jC for 2 days. In vitro methylation assay was performed as described by 2.3. Screening of a cDNA library by solid-phase methylation Najbauer et al. [16] with a few modifications. One micro- gram each of recombinant proteins was added in a 20 Al The HeLa UNI-ZAP cDNA expression library (Strata- reaction mix containing 50 mM Na-morpholinepropanesul- gene, CA) was plated on Escherichia coli XL-1 blue strain Fig. 1. Detection of methylated proteins by PRMT1 on a nitrocellulose membrane. (A) Nuclei extracts and cytosolic fractions prepared from HeLa cells cultivated for 3 days in either the absence (lanes 1 and 2) or presence (lanes 3, 4, 5, and 6) of the methyltransferase inhibitor adenosine dialdehyde (AdOX), blotted on a nitrocellulose membrane, and incubated with (lanes 1, 2, 3, and 4) or without (lanes 5 and 6) PRMT1. (B) Whole E. coli proteins from IPTG- induced GST-hnRNP A1 were separated on SDS-PAGE and transferred on a nitrocellulose membrane (lanes 2, 4 and 6). IPTG-untreated E. coli proteins were similarly prepared as negative controls (lanes 1, 3, and 5). The nitrocellulose membranes were incubated in the absence (lane 1 and 2) or presence (lanes 3 and 4) of recombinant PRMT1 in methylation reaction buffer including 3H-SAM at 30 jC for 1 h. The protein amount of each lane was normalized as shown in the Coomassie staining (lanes 5 and 6). K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 3 Fig. 2. Expression cloning protocol of substrate of PRMT1. (A) Brief protocol of the screening of PRMT1 substrates from a kgt 11-HeLa cDNA library. (B) Representative result of the first screening (upper panel). Positive clones indicated by arrows (I, II, and III of the upper panel) were further condensed to monoclonal plaques through a second (not shown) and third screening (lower panels). 4 at a density of 1.8 Â 10 plaques per 100 Â 140 mm square 10 mM isopropyl-h-D-thiogalactopyranoside [IPTG]. The agar plate. After incubation for 3 h at 42 jC, the plates were plates were incubated at 37 jC for another 4.5–5 h, and overlaid with nitrocellulose membrane filters (PROTRAN, their filters were peeled off, immersed in blocking solution Schleicher & Schuell, NH) that had been soaked with [5% skim milk in PBS] and gently agitated at 4 jC for 1 h. Table 1 Summary of expression screening for PRMT1 substrates 4 K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 The filters were then washed three times for 20 min in PBS, nant PRMT1 and further incubated at 30 jC for 2–3 h for incubated for 30 min at 4 jC in the methylation buffer (50 the solid-phase methylation with gentle agitation.

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