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J. Biol. Chem., Vol. 275, Issue 42, 32793-32799, October 20, 2000

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Novel RING Finger Proteins, Air1p and Air2p, Interact with Hmt1p and Inhibit the Arginine Methylation of Npl3p^*

Koichi Inoue, Takayuki Mizuno^§, Kazuhiro Wada, and Masatoshi Hagiwara^¶

From the  Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan and ^§ SEEDS Laboratory, Institute of Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan

Received for publication, May 26, 2000, and in revised form, June 29, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Various types of protein modifications, such as phosphorylation, acetylation, glycosylation, and myristoylation, are now recognized in a number of eukaryotic proteins, and their functional significance has been exhibited. A potential role of protein methylation as a post-translational modification in signal transduction, however, has not been explored with the same breadth and intensity that protein phosphorylation has enjoyed in recent years. The methyl group, donated by SAM,¹ is transferred to carboxyl groups or to the side chain nitrogens of the amino acid lysine, arginine, or histidine (1). Evidence for the post-translational methylation of arginine residues in proteins was first provided by the presence of radioactive species chromatographing at positions near that of arginine in acid hydrolysates of isolated calf thymus nuclei incubated with [methyl-¹⁴C]SAM (2). The products of the methylation reaction were determined to be N^G-monomethylarginine and N^G,N^G-dimethylarginine (3). Type I protein arginine methyltransferases methylate arginine residues to form asymmetric N^G,N^G-dimethylarginine residues, and type II enzymes catalyze the formation of symmetric N^G,N'^G-dimethylarginine residues. Genes encoding type I methyltransferases have been identified in yeast (4, 5), rats (6), and humans (7), although type II protein arginine methyltransferases have not been cloned. In addition to these classical members, a coactivator-associated arginine methyltransferase 1 (CARM1) was recently cloned as a binding protein to p160 coactivators that include SRC1, GRIP/TIF2, and pCIP/RAC3/ACTR/AIB1/TRAM1 by using the yeast two-hybrid system to screen a mouse embryo cDNA library (8).

Although histones and myelin basic proteins were initially used as substrates to determine the arginine methyltransferase activity, hnRNPs are now known to be more efficient for substrates (9). hnRNPs are abundant RNA-binding proteins, and more than 20 different hnRNPs, designated A-U, have been identified (10, 11). hnRNPs are associated with nascent polymerase II transcripts and involved in many steps of mRNA processing and nuclear transport (10-12). In Saccharomyces cerevisiae, temperature-sensitive mutants of one such protein, Npl3p, display defects in export of mRNA from the nucleus (13-15). Npl3p is a yeast homologue of the prototypical hnRNP, A1, which has two RNA recognition motifs of 90 residues each and an arginine-glycine-rich C-terminal domain termed the "RGG box" (10). In addition to the ability of RNA recognition motifs, the RGG box domain (or RGG domain) has been shown to mediate RNA-protein interactions (16, 17), and arginine methylation typically occurs within the regions with the RGG domain in hnRNP A1 and Npl3p (6, 9, 18). Several other potential substrates for the enzyme including fibrillarin and nucleolin have a similar RGG methylation consensus: (G/F)GGRGG(G/F) (19).

Although the significance of arginine methylation in the RGG domain has not been revealed fully, Shen et al. (20) recently reported that Npl3p cannot exit the nucleus efficiently in cells lacking a major arginine methyltransferase, Hmt1p/Rmt1p, and overexpression of Hmt1p enhances Npl3p export. The export of another RNA-binding protein known to shuttle from the nucleus to the cytoplasm, Hrp1p, is also impaired when HMT1 is missing (20, 21). Thus, the efficient export of at least two shuttling hnRNPs implicated in RNA processing and export requires arginine methylation in the RGG domain.

These findings gave us the idea that unidentified protein(s) may interact with the RGG domain in a methylation-dependent manner as a regulator of the hnRNP shuttling. In support of this possibility, Michael et al. (22) reported that a part of C-terminal RGG domain is sufficient for its nuclear export when hnRNP A1 shuttles from the nucleus to the cytoplasm. In this report, we performed yeast two-hybrid screening with the C-terminal region (amino acids 281-414) of Npl3p as a bait and isolated several clones.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Microorganisms and Plasmids-- The genotypes and sources of S. cerevisiae strains used in this study are listed in Table I. Yeast strains were grown in standard YPDA (1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose, and 0.004% adenine sulfate) or synthetic complete media lacking the appropriate amino acids (23). Sporulation medium was purchased from Bio 101. 

The plasmids used for yeast in this study are listed in Table II. A PCR product of 0.4 kb encoding amino acid residues 281-414 of Npl3p was inserted into the BamHI-PstI gap of pBTM116 (25), named pBTM116NPL3S. A 1.0-kb PCR-amplified fragment containing the Air2p-coding region was inserted into the NcoI-XhoI gap of pACTII (26), designated pACTIIAIR2. A PCR-amplified 1.1-kb fragment containing the Air1p-coding region was inserted into the BamHI-XhoI gap of pYES2 (Invitrogen), designated pYES2AIR1. Three genes described above were PCR-amplified from the genome of S. cerevisiae strain S288C. pCgURA3 and pCgTRP1, plasmids for gene disruption in yeast, were kindly provided by Arisawa (27).

pGEX-HMT1 was kindly supplied by Silver (4). The DNA sequences encoding amino acid residues 1-207, 56-207, and 56-348 of Hmt1p were PCR-amplified as BamHI-XhoI fragments by using pGEX-HMT1 as a template. These fragments were inserted into the BamHI-XhoI gap of pGEX5X-3 (Amersham Pharmacia Biotech) and designated pGEX-HMT11, 2, and 3, respectively. pET28HMT1 was constructed by inserting a PCR-amplified 1.1-kb EcoRI-NotI fragment from pGEX-HMT1 into the EcoRI-NotI gap of pET28a (Novagen). A 1.2-kb PCR product containing full-length Npl3p-coding sequence was inserted into the SalI-NotI gap of pGEX5X-3 and named pGEX-NPL3. A PCR-amplified 1.1-kb fragment containing the Air1p-coding region from the genome of S. cerevisiae strain S288C was inserted into the BamHI-XhoI gap of pET28a, designated pET28AIR1.

Two-hybrid Screening-- The yeast strain L40 was transformed with pBTM116NPL3S as a bait. After that, the strain was transformed with the library containing cDNA fragments fused to the GAL4 activation domain (28). In total, ~7.0 × 10⁵ transformants were obtained, in which about 200 colonies grew on synthetic complete plates lacking tryptophan, leucine, and histidine (SC-Trp-Leu-His). As for the colonies, plasmids were recovered and transformed into an Escherichia coli strain MC1066 (29). Using the strain, the activation domain plasmids from the library were isolated by their ability to complement an E. coli leuB mutation as a result of the presence of the plasmid-borne LEU2 gene. To confirm that transcriptional activation was dependent on the presence of both gene fusions, the isolated library plasmids were retransformed into the original L40 strain with either pBTM116NPL3S or pBTM116a, which expresses only the LexA DNA-binding domain.² In each case, the two transformants and the strain containing pBTM116NPL3S and pACTII were streaked on SC-Trp-Leu-His plates. The colonies, which grew with the pBTM116NPL3S and did not with pBTM116a on SC-Trp-Leu-His plates, were selected. The plasmids were recovered again and sequenced. Data base searches were performed by use of the Yeast Proteome Database, the Saccharomyces Genome Database, and the NCBI BLAST program.

Expression of Proteins in Bacteria-- E. coli DH5 carrying the pGEX fusion constructs was grown in Luria-Bertani medium at 37 °C and induced with 0.5 mM for 5-6 h at 30 °C. Bacteria were harvested by centrifugation and lysed in resuspension buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM PMSF, and 1% Triton X-100). The cells were ruptured by sonication on ice, using an ultrasonic disruptor (UD-201; TOMY). Sonication (five pulses of 1 min each) was carried out at 50% duty cycle. After centrifugation at 6,000 × g for 30 min at 4 °C, the supernatant was mixed with glutathione beads at 4 °C overnight. The protein-bound beads were washed repeatedly with the same resuspension buffer. GST fusion proteins were then eluted a few times with 30 mM reduced glutathione in the resuspension buffer and dialyzed overnight against dialysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 2 mM dithiothreitol, 0.5 mM PMSF, and 20% glycerol) at 4 °C. Proteins were analyzed by SDS-PAGE, and appropriate fractions were stored at 80 °C.

pET28 fusion constructs were transformed with BL21(DE3), and purification was performed in much the same manner as for the pGEX fusion proteins with the following differences. Resuspension buffer containing 20 mM imidazole was used during sonication or washing, and 30 mM reduced glutathione was replaced with 200 mM imidazole.

GST Pull-down Experiments-- For in vitro protein-protein interaction assay, 3 µg of GST or GST fusion proteins were incubated for 1.5 h at 4 °C with 10 µl of glutathione-Sepharose beads in the binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, and 1% Triton X-100). After washing with the binding buffer once, the beads were incubated for 6 h at 4 °C with 0.5 µg of T7-tagged proteins in the binding buffer. The beads were then washed five times with the binding buffer, and retained proteins were eluted with 30 mM reduced glutathione in the binding buffer.

Protein Analysis-- The aliquots of GST pull-down experiments were boiled in loading buffer and electrophoresed on sodium SDS-polyacrylamide gel. Separated proteins on SDS-PAGE were transferred to nitrocellulose membranes, which were then blocked in 5% dry milk in phosphate-buffered saline and probed with anti-GST rabbit serum (Z-5; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-T7 antibody (Novagen). After washing in phosphate-buffered saline five times, the membranes were probed with an alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse antibody (Zymed Laboratories Inc.), respectively. After washing with Tris-buffered saline with Tween 20 five times, proteins were visualized.

For the staining of HA-tagged proteins in yeast extracts, anti-HA polyclonal antibody (Medical and Biological Laboratories) was used.

Yeast Protein Extraction-- Yeast proteins were extracted as described (30). Each yeast strain was grown at 30 °C in galactose-containing synthetic medium. Cells were harvested to the optical density of 1.0 at 600 nm in the medium (1 ml), resuspended in 100 µl of sample buffer (60 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.0025% bromphenol blue), and heated immediately at 95 °C for 5 min. The extracted proteins were obtained in the supernatants following the centrifugation of the extracts, and the aliquots were analyzed.

Gene Disruptions-- Disruption of the HMT1 gene was performed as described (31). Briefly, two oligonucleotides were synthesized: olis146 and olis147 (see below). Oligonucleotide olis147 contains 50 nucleotides of the HMT1 5'-untranslated sequence followed by 20 nucleotides of the sequence from the 3'-end of CgURA3 (27). Oligonucleotide olis146 contains 50 nucleotides of the translated sequences of the HMT1 followed by 21 nucleotides from the 5'-end of CgURA3. The PCR products were amplified with pCgURA3 as a template and olis146 and olis147 as primers and transformed in ETM43. We selected Ura+ transformants, and the structure of the disrupted genes was verified by PCR.

To construct a null mutant of AIR1 or AIR2, we used two pairs of oligonucleotides, olis454-455 (see below) or olis451-452 (see below), as primers and pCgURA3 or pCgTRP1 as templates, respectively. Diploid strain W303 was transformed with the PCR products. Sporulation and tetrad analysis yielded a haploid strain containing air1::CgURA3 or air2::CgTRP1, designated ETM52 or ETM56, respectively. To create the parental strain of double disruption mutant air1::CgURA3 and air2::CgTRP1, ETM52 was mated with ETM56. The diploids were selected, tetrads were dissected, and Trp+/Ura+ spores were selected, as ETM159.

The primers used for gene disruption were olis146 (5'-CACCATATAATTCTCTCATTTTCTATTTTATACTGTATAATGAATCTTTTACAGCTATGACATGATTACG-3'), olis147 (5'-ATGAATGGAGCACTTGTCGGGAAAGATCAGACCGCCTTCTACCAAATAGTCCCAGTCACGACGTTGTAAAA-3'), olis451 (5'-TTATTACCATCAAATGTATTCATGATCCGGGTAGTGCCTTTTTTTCCTTCCCCAGTCACGACGTTGTAAAA-3'), olis452 (5'-GACCTGTAATGACCCACCTCATCACACTTTGAACACTGGATAGCCTTCGGACAGCTATGACATGATTACG-3'), olis454 (5'-AAGTGACTTCCATAGGCGCTTGTCCTTTGTCTTTGTTTCTTGCTACTGTTACCCAGTCACGACGTTGTAAAA-3'), and olis455 (5'-AAGCGTGCAGAACACCTTCTTCCATTTATGAGGACACTGTGATTTATAACAGCTATGACATGATTACG-3').

Fluorescent in Situ Hybridization-- Each strain was grown to midlog phase in YPDA at 30 °C. The cells were then subjected to in situ hybridization as described (32) with some modification. Fixation of the cells was done with freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 6.0) for 1 h at room temperature. After washing three times in 0.1 M sodium phosphate buffer, 10⁶ cells were treated with 2 µl of -mercaptoethanol and 20 µl of 2 mg/ml Zymolyase 100T (Seikagaku Corp.) in 200 µl of sorbitol buffer (1.2 M sorbitol, 0.1 M sodium phosphate buffer) for 20-30 min at 37 °C. Resultant spheroplasts were washed three times in ice-cold sorbitol buffer and were put into the wells of Teflon-faced multiwell slides (Nalge Nunc International) coated with poly-L-lysine (M_r 150,000-300,000; Sigma). After 30 min, nonadhered cells were removed by aspiration, and the slides were plunged into 70, 90, and 100% ethanol for 5 min successively. The slides were then dried completely to avoid condensation and incubated in prehybridization buffer containing 4× SSC, 5× Denhardt's solution, and 1 mg/ml tRNA at room temperature for 30 min. Hybridization was carried out with the same buffer containing 2.5 pM Cy3-labeled oligo(dT)₅₀ for 12-16 h at 42 °C in a moisture chamber. Following hybridization, cells were washed four times in 4× SSC at 42 °C for 1 h each and counterstained with 1 µg/ml of 4',6-diamido-2-phenylindole. The slides were mounted with a mounting medium (90% glycerol, 10% phosphate-buffered saline) and were examined under BX-50 (Olympus).

In Vitro Methylation Assay-- In vitro methylation assay was done as described (4) with some modification. 1 µg of GST-Npl3p was incubated at 30 °C for 45 min in a 20-µl reaction containing 50 mM Na-MOPS (pH 7.2), 300 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 10% glycerol, 0.5 µg of GST-Hmt1p, 0.4 mM [methyl-³H]SAM (specific activity of 1.3 Ci/mmol; NEN Life Science Products), and the indicated quantities of T7-Air1p or BSA (as a control). The reactions were terminated by adding SDS sample buffer and boiling for 5 min. 50% of each reaction solution was then loaded onto 15% SDS-polyacrylamide gel and subjected to electrophoresis. Gels were stained with Coomassie Blue R-250 to visualize protein bands and then fluorographed. The results represent 2-day exposures at 80 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Npl3p-interacting Proteins by Two-hybrid Screening-- We screened ~7 × 10⁵ transformants of yeast cDNA library using a bait of RGG domain of Npl3p (Fig. 1A) as described under "Experimental Procedures." The results yielded nine different genes summarized in Table III. One clone is, as expected, Hmt1p/Rmt1p, and seven clones encode RNA-interacting proteins. Moreover, five of seven have the regions with RGG repeats, the RGG domain. High frequencies of the RGG proteins can be explained by the interaction between the RGG domains, since Cartegni et al. (44) reported that hnRNP A1 proteins interact with each other with the RGG domain. The other clone is the YIL079 gene, which encodes an unknown open reading frame. According to the data of the Yeast Proteome Database, the open reading frame is 1080 nucleotides in length, encoding a basic (predicted pI = 9.2) protein of 360 amino acid residues with a predicted molecular mass of 41.647 kDa. Analysis of the predicted amino acid sequence of the gene product indicates that it contains the RING finger domain near the N terminus (45).

View larger version (53K): [in this window] [in a new window]   Fig. 1.   A, schematic representation of full-length Npl3p (top) or the C terminus of Npl3p (bottom), the latter of which is used as a bait in the two-hybrid screening. B, dot-spot analysis for comparison of interaction in wild-type host or hmt1-deprived host between Npl3p and the clones segregated in a two-hybrid assay. On each plate, the upper two spots represent interactions in wild-type host, and the lower two spots represent interactions in the hmt1-deprived host. Precultures were diluted in growth medium, and equivalent amounts of cells (diluted in 101 steps) were spotted onto synthetic complete medium plates lacking tryptophan and leucine or lacking tryptophan, leucine, and histidine. Plates were incubated for 3 days at 30 °C. In the clones, only YIL079 and SUB2 (a control for comparison with the case of YIL079) were shown.

Next, we examined the interaction of the listed proteins with Npl3p in an hmt1 disruptant as a host for two-hybrid system to test their Hmt1p-dependent interaction. The growth of the hmt1 disruptant was similar to or even faster than that of the wild-type strain on a synthetic complete plate lacking histidine, when eight of the nine clones were introduced as preys (Fig. 1B and data not shown), indicating their Hmt1p-independent interaction with Npl3p. In contrast, the hmt1-deprived strain introducing NPL3 (nuclear protein localization 3) and YIL079 could not survive in the medium lacking histidine, suggesting that the YIL079 gene product can interact with Npl3p only in the presence of Hmt1p (Fig. 1B).

YIL079 Gene Product Interacts with Hmt1p but Not with Npl3p in Vitro-- The observation shown in Fig. 1B that the interaction of Npl3p with the YIL079 gene product, Yil079p, depends on the presence of Hmt1p implies two possibilities: (i) Npl3p interacts with Yil079p mediated by Hmt1p, or (ii) the methylated state of Npl3p mediated by Hmt1p is required for interaction with Yil079p. To determine which is the truth, physical interactions of Hmt1p, Npl3p, and Yil079p were examined in vitro. As shown in Fig. 2A, T7-tagged Hmt1p bound to GST-Npl3p but not to GST alone (Fig. 2A, lanes 1 and 3). T7-tagged Yil079p, however, did not bind to GST-Npl3p (Fig. 2A, lane 4). Instead, this protein bound to GST-Hmt1p, but not to GST alone (Fig. 2A, lanes 2 and 5). Although we tried to detect the possibility of methylation-dependent interaction of Npl3p and Yil079p, we could not find any direct interaction between them even after methylation (data not shown). Thus, we designated the YIL079 gene product Air1p (arginine methyltransferase-interacting RING finger protein).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Air1p binds to Hmt1p in vitro. A, 0.5 µg of T7-tagged proteins were suspended in binding buffer and incubated with glutathione-Sepharose beads bound to 3 µg of GST or GST fusion proteins. The beads were washed, and T7-tagged proteins were analyzed by 15% SDS-PAGE and visualized. B, schematic representation of wild-type (WT) and deletion mutants of Hmt1p used in C. WT Hmt1p is the full-length 348-amino acid protein (4, 5). Shaded boxes (I, post I, II, and III) indicate conserved regions within most of the SAM-dependent methyltransferases (46). The numbers in parentheses indicate amino acids present in the deletion mutants. C, differential in vitro binding of Air1p to wild-type and deletion mutants of Hmt1p. The indicated GST fusion proteins (shown by the arrowheads) were immobilized on glutathione-Sepharose resin and analyzed for T7-Air1p binding as shown in A. The proteins were detected by Western blot analysis.

To identify the portion of Hmt1p required for binding to Air1p, we prepared deletion mutants of Hmt1p fused to GST (Fig. 2B). Hmt1p is one of the SAM-dependent methyltransferases, and motifs I, post-I, II, and III are conserved among most of them (46). The mutant constructs were analyzed in vitro in a GST pull-down assay as mentioned under "Experimental Procedures" (Fig. 2C). The lower panel of Fig. 2C shows GST fusion proteins stained with anti-GST rabbit serum, and the upper panel shows the precipitated T7-Air1p recognized with anti-T7 antibody. T7-Air1p was allowed to bind to wild type and 1 (residues 1-207), but not to 2 (residues 56-348) and 3 (residues 56-207), indicating that the N terminus of Hmt1p is essential for the binding to Air1p.

Air1p Inhibits Methylation of Npl3p Mediated by Hmt1p-- To examine the effect of Air1p on the methyltransferase activity of Hmt1p, GST-Hmt1p and GST-Npl3p were incubated with [methyl-³H]SAM with the indicated amount of recombinant T7-Air1p or BSA, and the status of arginine methylation of GST-Npl3p was examined. The methylation of Npl3p was inhibited by T7-Air1p in a dosage-dependent manner, whereas BSA did not affect the methylation of Npl3p (Fig. 3A).

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Inhibition of effect of Hmt1p by Air1p. A, inhibition of methylation status of Npl3p by Air1p in vitro. Purified GST-Npl3p (1 µg), GST-Hmt1p (1 µg), and 15 mM [methyl-3H]SAM were incubated (4) with T7-Air1p or BSA in the absence or presence of peptides at the indicated quantities. The methylation status of GST-Npl3p was evaluated by fluorography. B, inhibition of Hmt1p-dependent suppression of npl3 temperature-sensitive mutant. npl3-17 strain (13) was co-transformed with either pNB1HA-HMT1 or pNB1 (control vector) under the control of the ADH promoter and either pYES2AIR1 or pYES2 (control vector) under the control of the GAL1 promoter. C, effect of Air1p on the expression of Hmt1p. npl3-17 strains containing an HA-Hmt1p expression vector, pNB1HA-HMT1, were grown in the presence or absence of overexpression of Air1p. The same amounts of extract proteins from yeast transformants were resolved on SDS-PAGE, transferred to a nitrocellulose membrane, and visualized with anti-HA antibody.

Next, we examined whether Air1p would inhibit the function of Hmt1p even in vivo. The growth of an npl3 mutant strain, npl3-17, was arrested at the sensitive temperature (34.5 °C) (Fig. 3B). As Shen et al. (20) have reported, the growth defect of the npl3-17 strain was rescued by overexpression of Hmt1p, suggesting that hypermethylation by Hmt1p complements the function of mutated Npl3p. However, when Air1p was overexpressed with Hmt1p in the npl3-17 strain, the growth rescue by Hmt1p disappeared (Fig. 3B). Although we examined the effect of Air1p overexpression on the expression level of Hmt1p in an npl3-17 strain, the expression of Hmt1p was not affected by Air1p (Fig. 3C). These indicated that Air1p could antagonize the methylation of Npl3p by Hmt1p both in vitro and in vivo.

YDL175 Gene Product Is a Homologue of Air1p in S. cerevisiae-- Homology searches using BLAST 2.0 indicate that YDL175 gene product in S. cerevisiae has significant identity (45%) with Air1p (Fig. 4A). The gene product also has the RING finger domain, and a cysteine-rich region at the N terminus side including the RING finger domain is highly homologous to amino acid residues 32-217 of Air1p (68%). There were no other clearly homologous proteins in any species in our homology search. In two-hybrid system, cells that expressed Npl3p as a bait and the YDL175 gene product as a prey could grow up in the medium lacking histidine only in the presence of Hmt1p (Fig. 4, B and C), indicating that the interaction of the gene product with Npl3p requires Hmt1p. Indeed, the gene product also bound to Hmt1p in vitro (data not shown). So we designated it as Air2p.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Comparison between Air1p and Air2p. A, the amino acid sequence alignment of Air1p and Air2p. The positions of identical or similar amino acids between these two proteins are indicated by asterisks or dots, respectively. Gaps (-) were introduced to optimize the alignment, which was performed using a Lipman-Peason method algorithm and GENTYX-MAC version 10.0 software. The conserved cysteines or histidine in the RING finger domain are underlined. Amino acid positions are indicated on the left. B, two-hybrid interactions between Npl3p and Air2p. Npl3p+ or Npl3p indicates LexA fused to the C terminus of Npl3p or LexA only as bait, respectively. Air2p was fused in frame to the Gal4p activation domain in the vector pACTII (26). Individual transformants grown in synthetic complete medium plate lacking tryptophan and leucine (SC-WL) were selected and streaked on synthetic complete medium plates lacking tryptophan, leucine, and histidine (SC-WLH). C, alteration of the interaction between Npl3p and Air2p. Upper two spots stand for interaction in host with HMT1, and the lower spots show the interaction without HMT1 as shown in Fig. 1B.

Air1p and Air2p Have Functional Redundancy on Cell Growth-- To clarify the physiological role of Air1p and Air2p, we examined the growth of air1, air2, and air1 air2 disruptant strains. The air1::CgURA3 and air2::CgTRP1 constructs were targeted to the AIR1 and AIR2 chromosomal loci in the diploid W303 by homologous recombination, respectively, and correct replacements were verified by PCR (data not shown). After sporulation and tetrad dissection, the transformed diploid yielded four viable spores with normal growth from each tetrad, with two being prototrophic and two auxotrophic for uracil and tryptophan, respectively. Single disruption of either AIR1 or AIR2 had no effect on cell growth (Fig. 5). To test whether Air1p and Air2p act redundantly, we obtained the air1 air2 double disruptant. The diploid was constructed by air1 and air2 single disruptants, and tetrad analysis was conducted. Deletion of both AIR1 and AIR2 caused a strong growth defect of cells at 30 °C (Fig. 5).

View larger version (57K): [in this window] [in a new window]   Fig. 5.   Growth phenotype of wild-type strain, air1 or air2 single disruptant strains, and air1 air2 double disruptant strain. The strains with air1 or air2 single disruption and the air1 air2 double disruption were constructed as described under "Experimental Procedures." Below are shown double disruptants segregated independently. Cells were streaked and grown on YPDA plates at 30 °C for 2 days.

To find the reason for the growth defect of the double disruptants, we analyzed the subcellular localization of poly(A)⁺ RNA in the wild-type or mutant strains by in situ hybridization using Cy3-labeled oligo(dT)₅₀. However, while each single disruptant showed the similar cytosolic poly(A)⁺ RNA localization (Fig. 6, H and J) as that of wild type (Fig. 6B), clear nuclear accumulation of mRNA was observed in the air1 air2 double disruptants (Fig. 6E).

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Localization of poly(A)+ RNA in wild type and various disruptants. Cultures of W303-1A, ETM52, ETM56, and ETM159 grown to the midlog phase at 30 °C were prepared for hybridization with oligo(dT)50 as a probe as described under "Experimental Procedures." Then they were inspected by phase-contrast (A, D, G, and I) and fluorescence microscopy for detecting poly(A)+ RNA (B, E, H, and J). Nuclei were marked with 4',6-diamido-2-phenylindole (DAPI) (C and F). A-C display wild-type cells. G and H show air1 cells. I and J show air2 cells. D-F show air1 and air2 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Since the post-translational methylation of protein arginine residues was revealed (2), their activity has been researched (46). Type I protein arginine N-methyltransferases methylate protein arginine residues to form N^G,N^G-dimethylarginine residues (asymmetric). Hmt1p/Rmt1p was isolated from S. cerevisiae as a protein arginine methyltransferase that catalyzes both the N^G-monomeric and N^G,N^G-asymmetric dimethylation of arginine residues (4, 5). Hmt1p may be the one and only type I protein arginine N-methyltransferase present in the yeast, because the mutation of the gene of S. cerevisiae eliminates many N^G,N^G-dimethylarginine residues (5). In contrast to the yeast, at least two active type I arginine N-methyltransferases, PRMT1 and PRMT3, have been identified in mammals (6, 7, 47). One of the fundamental observations reported in recent years, as mentioned in the Introduction, is the possibility of facilitating mRNA export from the nucleus into the cytoplasm. In the process of the revelation of mRNA export, we cloned HMT1 and AIR1 using Npl3p as a bait in the two-hybrid screening. An in vitro binding assay showed that Air1p interacts with the RGG domain of Npl3p bridged by Hmt1p. McBride et al. (48) reported that the N-terminal E18V substitution of Hmt1p affected methylation of Npl3p but not of other substrates and suggested that Glu¹⁸ is involved in substrate binding or positioning. In support of this finding, the N-terminal deletion mutant of Hmt1p used in this study, 2 (residues 56-348), could not methylate Npl3p in vitro (data not shown).

The inhibitory effect of Air1p on the enzyme activity of Hmt1p was detected by in vitro methylation assay of Npl3p. The methylation of Npl3p is blocked by Air1p in a dose-dependent manner, and overexpression of Air1p inhibited the Hmt1p-dependent growth in vivo. In this case, it is possible that Air1p suppresses the expression of Hmt1p followed by the elimination of the rescue by Hmt1p in an npl3-17 strain. However, since overproduction of Air1p did not affect the amount of exogenous Hmt1p introduced by a high copy plasmid, the suppression of Hmt1p-dependent rescue by overproduction of Air1p is not due to the reduced expression of Hmt1p. Hmt1p has been shown to facilitate the export of Npl3p in nucleocytoplasmic transport (20). Our observation that growth and mRNA transport were affected in cells lacking AIR1 and AIR2 suggests to us that the regulated methylation of Npl3p may be crucial for mRNA transport. When we checked the localization of the GFP-Npl3p in wild type and air1 air2 double disruptant, GFP-Npl3p was localized in the nucleus in both strains, and any difference of the expression pattern was not observed (data not shown). The regulatory mechanism of Air1p and Air2p on mRNA transport should be further elucidated.

It should be noted that Liu and Dreyfuss (9) reported that arginine methylation was not observed in yeast extracts when recombinant hnRNP A1 was used as a substrate, the contrary to the extensive in vivo methylation of hnRNPs in HeLa cells. The inconsistent observations could be explained by the presence of endogenous inhibitory factors like Air1p and Air2p in yeast. An unidentified signal transduction system could be involved in the regulation of protein methylation mediated by Hmt1p as suggested in mammalian cells (1). PRMT1 has been reported to interact with interferon receptor and to affect the proliferation induced by interferon (49). PRMT1 also interacts with TIS21 and BTG1, which are immediate early genes and have an antiproliferative effect, and the methylation activity was modulated by the interaction (6).

Air1p and Air2p have sequence similarity, especially in their N-terminal RING finger domain. The RING finger domain consisting of the amino acid sequence Cys-X₂-Cys-X_9-39-Cys-X_1-3-His_2-3-Cys-X₂-Cys-X_4-48-Cys-X₂-Cys is a cysteine-rich zinc-binding domain ubiquitous in species ranging from plants to mammals and has been found in several proteins, which have important physiological roles in various biological phenomena (44). The RING finger domain is now believed to be a motif for protein-protein interaction. We could not detect any homologous proteins in data bases of other species, but some proteins have conserved cysteine and histidine over the RING finger domain. Identification of the mammalian homologues of Air1p or Air2p may pave the way to understand the signal-mediated regulation of methyltransferases in mammals.

	ACKNOWLEDGEMENTS

We thank P. A. Silver for the npl3-17 strain and plasmid pGEX-HMT1 and for the expression vector of GFP-Npl3p; M. Arisawa for plasmids of yeast selection markers; N. Hayashi for the yeast cDNA library for two-hybrid screening; Y. Mukai and S. Harashima for S. cerevisiae strains and plasmids; T. Tani, E. Noguchi, T. Zama, and J. Nakabayashi for useful technical comments; and H. Nakayama and S. Hashimoto for helpful comments while preparing the manuscript.

	FOOTNOTES

^* A part of this work was performed in the SEEDS Laboratory of Yamanouchi Pharmaceutical Co., Ltd. as a part of the research and development project of the Industrial Science and Technology Program supported by NEDO (New Energy and Industrial Technology Development). This work was supported in part by the Research for the Future Program, the Inamori Foundation, Nissan Science Foundation, the Mitsubishi Foundation, and Grants from the Ministry of Education, Science and Culture in Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. E-mail: m.hagiwara.end@mri.tmd.ac.jp.

Published, JBC Papers in Press, July 13, 2000, DOI 10.1074/jbc.M004560200

² Y. Mukai, unpublished results.

	ABBREVIATIONS

The abbreviations used are: SAM, S-adenosyl-L-methionine; hnRNP, heterogeneous nuclear ribonucleoprotein; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; GFP, green fluorescent protein; PCR, polymerase chain reaction; kb, kilobase(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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