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Biochimica et Biophysica Acta 1591 (2002) 1–10 www.bba-direct.com

Identification of methylated proteins by protein arginine N- 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- (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- , 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. The filters mM Na-MOPS [pH 7.2], 2 mM EDTA, 0.3 M NaCl, 0.1 Ag/ were then washed with TPBS three times for 20 min and air ml leupeptin, 1 mM PMSF, 0.4 AM 3H-SAM; specific dried. The filters were fluorographed with EN3HANCE activity of 1.3 Ci/mmol) with 0.02–0.04 Ag/Al of recombi- spray (Dupont, DE), dried and exposed to X ray film

Fig. 3. Primary structure and expression of SAMT1. (A) Predicted amino acid sequence of SAMT1. RGG motifs are indicated by black lines. (B) Expression of SAMT1 in various human tissues. Human multiple tissue Northern blot I or II (Clontech) was hybridized with a probe of full-length SAMT1 cDNA. The tissues from which the RNA is derived are indicated above and size standards (in kb) are indicated to the left. K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 5

Fig. 3 (continued).

(BioMax MR-2, Kodak, NY) at À 80 jC for 1 week. At the room temperature, permeabilized, and blocked in PBS con- first screening, 607 positive clones were detected in taining 0.4% Triton X-100, 1.5% BSA, and 5% normal goat 2.0 Â 105 plaques. We picked up 100 of these clones and serum. succeeded to isolate 83 positive clones by the third screen- ing. Positive clones were converted to plasmids with the Exassist/Solr E. coli system (Stratagene). 3. Results

2.4. Northern hybridization analysis 3.1. Detection of methylated proteins by PRMT1 on a nitrocellulose membrane SAMT full-length cDNA probe or h-actin probe was labeled with radioactive [a-32P] dCTP using the multi- First we examined whether proteins in HeLa cell prime DNA labeling system (Amersham Biosciences). A extracts could be methylated by PRMT1 on a nitro- human multiple tissue Northern blot (Clontech, CA) cellulose membrane. HeLa cells were cultivated for 3 membranes were used. Hybridization was carried out at days in the presence of the methyltransferase inhibitor, 20 42 jC overnight in hybridization mixture (6 Â SSC [1 Â AM adenosine dialdehyde (AdOX), to prevent endoge- SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50% nous methylation that would mask our assay. Nuclei and formamide, 1% SDS, 1 Â Denhardt’s solution, 10% cytosolic fractions were then size-separated on SDS- dextran sulfate, 100 Ag of denatured salmon sperm DNA PAGE, transferred to a nitrocellulose membrane, and per ml). The membrane was washed three times at room incubated with recombinant human PRMT1 and the temperature in washing buffer (0.1 Â SSC, 0.1% SDS), methylation substrate 3H-SAM at 30 jC for 30 min. and the hybridized transcripts were observed with a Four major bands and seven minor bands in the nuclei BAS2000 image analyzer (Fuji Film, Tokyo). fraction appeared after 2 days exposure (Fig. 1A, lane 3), whereas four major bands and three minor bands 2.5. Cell culture and transfection assays appeared in the cytosolic fraction (Fig. 1A, lane 4). All these bands were faint or absent in extracts prepared HeLa cells were grown on cover slips in Dulbecco’s from HeLa cells cultivated without AdOX (Fig. 1A, lanes modified Eagle’s medium [DMEM] supplemented with 1 and 2), suggesting that most arginines in these proteins 10% calf serum, 100 U/ml penicillin, and 100 U/ml were already methylated in vivo in HeLa cells. To further streptomycin. Expression vectors of pGFP (Clontech) examine the validity of the in vitro methylation reaction containing cloned full-length cDNAs were introduced into on the nitrocellulose membrane, we methylated recombi- cells at approximately 60% confluence with Lipofect- nant hnRNP A1 under the control of an IPTG promoter AMINE (Life Technologies, NY) following the trans- in E. coli. Proteins were isolated from IPTG-treated or fection conditions as recommended by the manufacturer. -untreated whole E. coli containing recombinant hnRNAP For immunoflourescence, cells were washed in PBS and A1, separated on SDS-PAGE and transferred to a nitro- fixed for 15 min with 4% paraformaldehyde in PBS at cellulose membrane. Methylated proteins were specifically 6 K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 detected in the IPTG-induced E. coli containing hnRNP RGG motifs at their C-terminus (indicated by stripes in A1 (Fig. 1B, lanes 3 and 4).InE. coli not containing the right end column of Table 1). Eight clones encoded human hnRNP A1, no positive band was found in IPTG- the same novel protein, which also has RGG motifs at its treated E. coli (Fig. 1B, lanes 1 and 2). These results C-terminus, but not other RNA-binding motifs. We show in vitro methylation by PRMT1 is specific to the termed this clone SAMT1 (substrate of arginine methyl- human protein and that E. coli does not methylate the transferase 1). The predicted amino acid sequence of human protein, at least at the proprietorial arginine SAMT1 is shown in Fig. 3A. During preparation of this residues. These results prompted us to perform a com- manuscript, a clone named CG1-55 coding the identical prehensive substrate screening of PRMT1 using a Egt11- protein to SAMT1 was registered to the database of HeLa cDNA expression library in E. coli host with GeneBank, as one of conserved human by com- IPTG-induced cDNA protein products separated on the parative proteomics [18]. Northern blot analysis of nitrocellulose membrane. SAMT1 mRNA showed ubiquitous expression in human tissue samples (Fig. 3B). We tagged cloned cDNAs with 3.2. Expression screening of PRMT1 substrates green fluorescent protein (GFP) and GFP-tagged SAMT1 was predominantly localized to the cytoplasm around Fig. 2 shows the experimental order of the screening nucleus, presumably at the endoplasmic reticulum (ER), (Fig. 2A) and a representative of the first and third while the other RGG-proteins were localized in the screening (Fig. 2B). After the first screening, 607 positive nucleus in HeLa cells (Fig. 4). Other five clones encode clones were detected in 2.0 Â 105 plaques. We picked up a transcription factor, ZF5 (zinc finger 5) and a GPI 100 of these clones and succeeded to isolate 83 positive (glycosyl phosphatidylinositol)-anchor protein, p137GPI. clones by the third screening. We determined the nucleo- Both of these proteins do not have typical RGG motifs. tide sequences of 48 of these clones. A BLAST search [17] against GeneBank revealed that more than half of 3.3. Methylation of cloned proteins by PRMT1 in vitro the clones encode putative RNA-binding proteins such as hnRNP R, hnRNP K, GRY-RBP (glycine, arginine, and To determine whether the cloned proteins are really tyrosine-rich RNA binding protein), TAFII68 (TBP asso- methylated by PRMT1 in vitro, we prepared recombinant ciated factor II68), RBP56 (RNA binding protein 56), and proteins of GST (glutathione sulfatransferase)-ZF5, GST- TLS/FUS (translocated in liposarcoma), as summarized in p137GPI, GST-hnRNP R, GST-GRY-RBP, GST-TAFII68, Table 1. A common feature of these proteins is to contain GST-TLS, and GST-SAMT1 (Fig. 5A). These proteins

Fig. 4. Intracellular localization of cloned proteins. HeLa cells were transfected with the expression vectors, fixed, and analyzed by indirect immunofluorescence staining. PRMT1 was expressed as a fusion protein with the HA epitope and stained with an anti-HA monoclonal (a). The cloned RGG proteins, hnRNP

R (b), GRY-RBP (c), TLS (d), TAFII68 (e), and SAMT1 (f) were tagged with GFP protein and stained with anti-GFP monoclonal antibody. Fluorescein isothiocyanate-labeled anti-mouse second antibody was used to visualize each protein. Scale bar in (a): 20 Am. K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 7

Fig. 5. Methylation of cloned proteins by PRMT1 in vitro. (A) Recombinant proteins of GST-hnRNP R (lane 1), GST-GRY-RBP (lane 2), GST-TAFII68 (lane 3), GST-TLS (lane 4), GST-SAMT (lane 5), GST-ZF5 (lane 6), and GST-GPI anchor protein (lane 7) were purified from E. coli and incubated with [methyl3H]-SAM in the presence of recombinant PRMT1. GST was used as a negative control (lane 8). Methylation of each protein was detected by autoradiography (upper panels) and protein amount of each methylation reaction was confirmed with Coomassie staining (arrow heads of lower panels). (B) The 63-VLD-65 to AAA mutation of 3 PRMT1. (C) GST-hnRNPA1, GST-TAFII68 and GST-SAMT1 were incubated with HeLa cell extract, H-SAM, and indicated amount of PRMT1 VLD to AAA mutant at 30 jC for 1 h. The amount of the RGG proteins were normalized by Coomassie staining as shown in the lower panels.

were then incubated at 37 jC for 30 min with recombi- GST-TAFII68, and GST-SAMT1 by the extract (Fig. 5C), nant PRMT1 and 3H-SAM. All of above proteins were suggesting that these proteins are methylated mainly by highly methylated by PRMT1, while GST alone was not PRMT1 in the HeLa cell extract. methylated (Fig. 5A). These proteins were methylated when they were incubated with HeLa cell extracts and 3H- SAM (Fig. 5C). Next, we tested the effect of a dominant- 4. Discussion negative PRMT1 on methylation of HeLa cell extracts. We mutated PRMT1 cDNA by substituting alanines for three Protein function is often modulated by posttranslational amino acids, valine 63, leucine 64, and aspartic acid 65, modifications; phosphorylation, acetylation, glycosylation, located in the highly conserved region that is proposed to be and methylation. Though an important role for protein important for SAM binding and thus for methytlransferase methylation in bacterial chemotaxis has long been appreci- activity [10] (Fig. 5B). The VLD to AAA mutation of ated (reviewed in Ref. [19]), a role of protein methylation in PRMT1 abolished the enzymatic ability to methylate hnRNP signal transduction has not been explored in mammalian A1 in vitro (data not shown). Administration of the mutant cells with the same breadth and intensity that protein PRMT1 suppressed the methylation of GST-hnRNP A1, phosphorylation has enjoyed in recent years. Unlike phos- 8 K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 phorylation, protein methylation might be irreversible, the other two do not have the consensus motif. Recently, because protein demethylases have not been discovered so Smith et al. [25] reported that most of the methylated far [20]. Yet, this may be a key mechanism of cell function arginine residues were found in Arg-X amino acid-Arg as a growing number of proteins are being found to contain (RXR) clusters in the C-terminus of PAB2. They showed methylated amino acids, monomethylarginine and asymmet- both PRMT1 and PRMT3 methylated the arginine in the C- ric dimethylarginine. Many of these proteins are associated terminal domain. As shown in Table 2, ZF5 and p137GPI with RNA, such as the heterogeneous nuclear ribonucleo- contained the RXR sequence, though the methylation sites protein (hnRNP) A1 [21,22], the U3 small nuclear RNA- of these proteins have not been identified yet. binding protein fibrillarin [23], the rRNA binding protein The glycine-rich clusters with many glycine–glycine nucleolin [24], poly(A)-binding protein (PAB) II [25], the dipeptides interspersed with aromatic or arginine residues Src-associated substrate, Sam68 [26], interleukin enhancer are termed ‘‘RGG’’ [31]. The RGG motif is ubiquitous in binding factor3 [27], and the yeast Npl3 protein [28,29].As proteins involved in diverse aspects of RNA metabolism a result, the hnRNPs contain about 65% of the total NG,NG- such as pre-rRNA processing factors, pre-mRNA splicing dimethylarginine found in the HeLa cell nucleus [11]. factors, hnRNPs, and RNA helicases [32]. The C-terminal The site of arginine methylation for type I enzymes RGG motif of hnRNP U, which lacks RRM (RNA recog- typically occurs within glycine- and arginine-rich (GAR) nition motif), facilitate as the RNA-binding motif [31].In domains from which similar consensus sequences have been nucleolin, which has two RRMs, the C-terminal RGG derived [4,7,16,30]. A search of the GenPept database for a domain mediates RNA binding even without the RRMs consensus FGGRGGF motif revealed 13 substrate candi- [31]. However, Bouvet et al. [33] reported that this domain dates [16]. Of these, 10 were known or presumed to interact interacts with 10 ribosomal proteins. Likewise, in hnRNP with RNA [16]. In our expression screening, we did not A1, RGG domain is not only involved in interaction with isolate any of 13 candidates, and seven of cloned 9 genes RNA [34] but also with proteins [35]. In spite of prevalence encode hnRNP R, hnRNP K, GRY-RBP, TAFII68, RBP58, of RNA binding proteins with RGG motifs, few studies TLS/FUS, and SAMT1, respectively, all of which contain have directly addressed the effect of arginine methylation on the consensus sequence or consensus-like sequences with binding of target proteins to RNA. However, recent studies two to three amino acid substitutions (Table 2). However, have examined arginine methylation in modulating protein–

Table 2 (F/G)GGRGG(F/G) homologous sequences and RXR sequences found in cloned proteins Gene (F/G)GGRGG(F/G) homologous RXR sequence Gene (F/G)GGRGG(F/G) homologous RXR sequence sequence sequence hnRNP R 447aa-GRGRGGGRGGY-458aa 106aa-RQR-108aa RBP58 203aa-GGDRGGF-209aa 185aa-RGR-187aa 500aa-RGRGGGRGGRG-509aa 306aa-RRR-308aa 334aa-RRGRGGYRGRGGFQ- 395aa-RGR-397aa 540aa-RGSRGGRGGP-549aa 516aa-RGR-518aa GRGGD-352aa 562aa-RGNRGGN-568aa 524aa-RGR-526aa 405aa-YRGRGGRGGDRGGY-418aa 554aa-RGR-556aa 456aa-GGDRGGG-462aa GRY-RBP 445aa-GRGRGGRGGY-455aa 103aa-RQR-105aa 464aa-GGDRGGG-470aa 495aa-ARGRGGR-501aa 303aa-RRR-305aa 472aa-GGDRGGG-478aa 536aa-RGARGGA-541aa 510aa-RGR-512aa 480aa-GGDRGGY-486aa 551aa-RGARGGRGGN-560aa 518aa-RGR-520aa 487aa-GGDRGGG-493aa 556aa-RGR-558aa 495aa-GGDRGGY-501aa hnRNP K 255aa-MRGRGGF-261aa 35aa-RSR-37aa 502aa-GGDRGGY-508aa 296aa-RGGRGGS-302aa 326aa-RGR-328aa 509aa-GGDRGGY-515aa TAFII 68 200aa-GGDRGGF-206aa 182aa-RGR-184aa 516aa-GGDRGGY-522aa 331aa-RRGRGGYRGRGGF-343aa 392aa-RGR-394aa 532aa-GGDRGGG-538aa 400aa-GYRGRGGRGGDRG- 523aa-RSR-525aa 559aa-GGDRGGG-565aa GYGG-417aa 567aa-GGDRGGY-573aa 453aa-GGDRGGG-459aa TLS/FUS 241aa-PRGRGGGRGGRGGM-254aa 216aa-RGR-218aa 461aa-GGDRGGG-467aa 380aa-GNGRGGRGRGGP-319aa 469aa-GGDRGGG-475aa 404aa-GGGRGGF-410aa 467aa-GGDRGGY-483aa 473aa-RGGRGGY-479aa 484aa-GGDRGGG-490aa 484aa-YRGRGGDRGGFRGGRGG- 492aa-GGDRGGY-498aa GDRGGF-506aa 499aa-GGDRGGY-505aa SAMT1/CGI-55 162aa-IRGRGGL-168aa 134aa-RER-136aa 506aa-GGDRGGY-512aa 169aa-GRGRGGR-175aa 513aa-GGDRGGY-519aa 364aa-RPGRGGRGGRGGRGR- 528aa-GGDRGGG-535aa GGR-382aa 556aa-GGDRGGG-562aa Zinc finger 5 ( À ) 291aa-RLR-293aa 564aa-GGDRGGY-570aa 382aa-RHR-384aa p137GPI ( À ) 303aa-RQR-305aa K. 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