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Genes to Cells (2009) 14, 309-317. doi:10.1111/j.1365-2443.2008.01266.x
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Nucleo-cytoplasmic shuttling of protein arginine methyltransferase 1 (PRMT1) requires enzymatic activity

 Frank Herrmann1 and Frank O. Fackelmayer2,*

1 EMBL-CRG Systems Biology, c/Doctor Aiguader 88, 08003 Barcelona, Spain
2 Biomedical Research Institute, Foundation for Research and Technology Hellas, 45110 Ioannina, Greece


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Methylation of arginine residues is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMT), of which PRMT1 is the predominant member in human cells. We have previously described the localization and mobility of PRMT1 in live cells, and found that it shuttles between the nucleus and the cytoplasm depending on the methylation status of substrate proteins. Recently, amino-terminal splicing isoforms of PRMT1 were shown to differ significantly in intracellular localization, the most interesting being splice variant 2 that carries a nuclear export signal in its amino terminus, and is expressed in increased levels in breast cancer cells. We show here that enzymatic activity is required for nucleo-cytoplasmic shuttling of PRMT1v2, as a catalytically inactive mutant highly accumulates in the nucleus and displays altered intranuclear mobility as determined by fluorescence recovery after photobleaching experiments. Our results indicate that nuclear export of PRMT1v2 is dominant over activity-independent nuclear import, but can only occur after activity-dependent release of the enzyme from substrates, suggesting that shuttling of the enzyme provides a dynamic mechanism for the regulation of substrate methylation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Post-translational modification of proteins is a dynamic mechanism that allows cells to rapidly react to changes in extra- and intracellular conditions without the need for synthesis of new proteins. Consequently, they are involved in processes as diverse as signal transduction, protein trafficking, transcriptional regulation, cell proliferation, and differentiation. One widespread post-translational modification is the methylation of arginine residues in proteins, that acts as an important regulator of protein function, and might be involved in protecting long-lived proteins from non-enzymatic aging (Bedford & Richard 2005; Fackelmayer 2005b). The modification is catalyzed by a small family of enzymes, the protein arginine methyl transferases (PRMT), of which eight members are known to date in humans (Bedford & Richard 2005). An additional enzyme that catalyzes arginine methylation has recently been described and termed FBXO11/PRMT9, but is structurally distinct from the eight canonic PRMTs and appears to have evolved independently from the PRMT family (Cook et al. 2006). In plants, PRMT10 and PRMT11 have been described, which are true members of the PRMT family but have no known human homologs (Niu et al. 2007; Scebba et al. 2007). Even though much effort has been put into research on arginine methylation, our knowledge of the exact biological role of the modification is still at its infancy, and more work on the cell biology and relevance for human disease is needed. Individual members of the PRMT family differ in abundance, specific activity, substrate specificity and intracellular localization, indicating that they serve non-redundant roles in vivo. The predominant member of the PRMT family, PRMT1, is responsible for at least 85% of all arginine methylation reactions in human and mouse cells (Pawlak et al. 2000, 2002; Tang et al. 2000). PRMT1 is an essential enzyme, because embryos from prmt1–/– knockout mice die shortly after implantation; however, as ES cells from such embryos are viable under cell culture conditions, the enzyme is apparently dispensable for basic cellular reactions such as gene expression and DNA replication (Pawlak et al. 2000). The major substrates of PRMT1 were identified as nuclear proteins interacting with nucleic acids, such as the core histones H3 and H4, components of the hnRNP particle involved in pre-mRNA processing and transport, or nucleolar components such as fibrillarin and nucleolin that are involved in pre-rRNA processing and ribosome biogenesis (Liu & Dreyfuss 1995; Wada et al. 2002; Herrmann et al. 2004; and references in Bedford & Richard 2005).

We have previously reported that PRMT1 is localized preferentially in the cytoplasm of human embryonic kidney cells, spatially separated from its major substrate proteins that reside in the cell nucleus. Interestingly, we found that the enzyme accumulates in the nucleus when methylation is inhibited by periodate-oxidized adenosine (Adox), and is tethered to unmethylated substrate proteins unless inhibition of methylation is terminated by removal of Adox (Herrmann et al. 2005). These results indicated, for the first time, that PRMT1 can shuttle between the cytoplasm and the nucleus, which probably contributes to the regulation of the enzyme. However, at the time of our original publication, we did not understand how nuclear-cytoplasmic shuttling is accomplished. More recently, elegant work from the laboratory of Jocelyn Coté has revealed the existence of seven alternative splicing isoforms that differ in their amino terminus (Goulet et al. 2007). Most interestingly, the splicing variant 2, which we had used for our localization and mobility studies, turned out to contain a nuclear export signal in the amino-terminus that leads to a predominantly cytoplasmic localization of PRMT1v2, fully confirming our earlier results. Even more interestingly, the authors found that the relative expression ratio of the isoforms varied significantly between normal and breast cancer cell lines, most prominent being an increase of the ratio of variant 2 over variant 1 mRNAs by an average factor of 3.5-fold. The altered PRMT1 isoform expression profile correlated with a differential pattern of arginine methylation in breast cancer versus normal cells (Goulet et al. 2007), as judged by immunolabeling with an antibody that exhibits excellent specificity towards asymmetrically dimethylated proteins (Boisvert et al. 2003). Given the role of methylation in epigenetic regulation of gene expression, the authors speculate that mis-regulation of arginine methylation could contribute to the onset of breast cancer.

Thus, variant 2 of PRMT1 appears to be an especially interesting splicing isoform of PRMT1, because it shuttles between the nucleus and the cytoplasm, and is up-regulated in breast cancer concomitantly with a change in substrate methylation. Based on our earlier work which showed that localization of PRMT1v2 changes dynamically in response to the methylation status of the cell (Herrmann et al. 2005), we have now investigated whether enzymatic activity contributes to intracellular shuttling of the enzyme. We show that an enzymatically dead PRMT1v2 strongly accumulates in the cell nucleus, where it forms a granular pattern, and is partially immobilized as determined by fluorescence recovery after photobleaching (FRAP) experiments. Finally, we find that the catalytically inactive mutant of PRMT1v2 forms hetero-oligomers with endogenous PRMT1, and cannot be expressed in high levels, suggesting that it acts as a dominant negative protein in vivo.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
In this article, we have investigated the role of enzymatic activity for the nucleo-cytoplasmic shuttling of PRMT1v2 in live cells. We used site-directed mutagenesis to introduce a three-residue change in the binding region for the methyl-donor S-adenosyl methionine. This mutation, changing the tripeptide VLD (residues 68–70) to three alanines, had previously been well characterized in vitro and results in a complete loss of enzymatic activity of PRMT1 (Wada et al. 2002) and the related PRMT4/CARM1 (Lee et al. 2002; Xu et al. 2004; Higashimoto et al. 2007). To analyze the potential effects of this mutation in vivo, we created cell lines stably expressing wild-type and mutant PRMT1 by selecting with G418 for 4 weeks. After selection, we characterized the mutant and the wild-type proteins by immunoprecipitation, and assayed for methylation activity. We found that both the wild-type protein and the mutant interact with the endogenous enzyme (Fig. 1B), even though the immunopreciptiation was carried out under stringent conditions using RIPA buffer. These conditions had earlier been shown to break the interaction of an identical mutant CARM1/PRMT4 with endogenous CARM1/PRMT4 (Lee et al. 2002). This result suggests that the mutual binding of PRMT1 monomers in the active complex is stronger than in the case of CARM1/PRMT4. Consequently, the immunopreciptated complex, also from cells expressing the mutant, had clearly detectable enzymatic activity (Fig. 1C), probably due to the molecules of endogenous, active PRMT1 in the complex. This also shows that, although PRMT1 must oligomerize to be enzymatically active (Zhang & Cheng 2003; Lim et al. 2005), the complex can tolerate a certain proportion of inactive enzyme. In these experiments we noticed, however, that mutant PRMT1 was expressed in significantly lower amounts than wild-type protein (Fig. 1B). The difference of expression was quantified by fluorescence assisted cell sorting, and revealed that mutant PRMT1 was expressed in approximately 10-fold lower amounts in comparison to wild-type PRMT1 (Fig. 2).


Figure 1
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  		Figure 1  Characterization of mutant PRMT1v2. Human embryonic kidney (HEK293) cells were transiently transfected (A) with wild-type PRMT1v2 or the catalytically inactive mutant VLD-AAA. Total cell extracts were prepared and subjected to western blotting with two independent anti-PRMT1 antibodies, right ab A33, left ab F339. (B) After 4 weeks of selection for stably expressing cell lines, total cell extracts were prepared and subjected to immunoprecipitation with monoclonal anti-GFP antibodies or an unrelated monoclonal antibody (control). Precipitated proteins were separated on an SDS gel and analyzed by western blotting with antibody 07-404 specific for PRMT1, or (C) tested for methylation activity using hypomethylated cell extract and radioactive methyl donor S-adenosyl-methionine; methylated proteins were detected by fluorography.

 

Figure 2
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  		Figure 2  Expression of the catalytically inactive mutant VLD->AAA is significantly lower than that of wild-type PRMT1v2. Stably transfected HEK293 cells as described in Fig. 1 were separated by fluorescence assisted cell sorting (FACS) into expressing and non-expressing cells after 4 weeks of G418 selection. Ten thousand cells per experiment were counted, and their individual fluorescence intensity monitored. Both populations contain comparable ratios of non-fluorescent cells (light line, area not filled) to fluorescent cells (heavy line, grey area), but fluorescence intensity of wild-type PRMT1 expressing cells is approximately ten times higher (peak at fluorescence intensity 1.1 x 104 vs. 1.1 x 103) than that of mutant PRMT1.

 
For a more detailed analysis of mutant PRMT1, we used fluorescent assisted cell sorting to obtain populations with equal protein expression level for wild-type and mutant PRMT1. Microscopic analysis (Fig. 3) revealed that wild-type PRMT1v2 was predominantly cytoplasmic, in line with our original observations (Herrmann et al. 2005) and the study of Goulet et al. (2007). For quantification, we analyzed more than 100 cells each of wild-type and mutant PRMT1 by measuring mean pixel intensities in several rectangular regions of interest. For each cell, the fluorescence intensity was determined both in the nucleus and in the cytoplasm, and divided to obtain the nuclear : cytoplasmic ratio. For wild-type PRMT1, this ratio was 0.18 ± 0.05 (translating to a sixfold difference in abundance), confirming earlier data (Herrmann et al. 2005). In contrast, mutant PRMT1 was strongly enriched in the cell nucleus, with a nuclear : cytoplasmic ratio of 1.48 ± 0.5. This difference between wild-type and mutant was more than eightfold, and statistically highly significant (P < 0.000005). We conclude that the catalytically inactive mutant is highly enriched in the nucleus despite its nuclear export signal, suggesting that it is tethered to nuclear substructures and unavailable for export.


Figure 3
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  		Figure 3  Catalytically inactive PRMT1 accumulates in the nucleus. Cells stably expressing equal amounts of wild-type or mutant PRMT1 were analyzed and quantified by confocal microscopy. Microscopic fields of typical cells (confocal slices) are shown, together with a quantification of the accumulation. For quantification, mean intensities in several regions of interest in the cytoplasm and the nucleus of more than 100 individual cells were measured, and expressed as a ratio of nuclear versus cytoplasmic fluorescence. Note that catalytically inactive PRMT1 accumulates in the nucleus approximately eightfold in comparison to wild-type PRMT1. Scale bar: 10 µm.

 
To address this point, we tested the interaction of wild-type and mutant PRMT1 with chromatin and the protein SAF-A that is present in a low-mobility skeletal structure of the nucleus (compare Fackelmayer 2005a). In the first experiment shown in Fig. 4A, we prepared total cell extracts by disrupting cells expressing wild-type or mutant PRMT1, and added MgCl2 to a concentration of 15 mM to precipitate chromatin. Under these conditions, significantly more mutant than wild-type PRMT1 was found in the chromatin precipitate, and was, consequently, reduced in the respective supernatant. In a second set of experiments, wild-type and mutant PRMT1 were immunoprecipitated, and the precipitates were assessed for coprecipitated histone H3 and SAF-A. We had shown previously that both proteins form stable complexes when methylation is chemically inhibited with Adox. Fully in line with these results, our immunoprecipitation assays show that histone H3 and SAF-A are detected in the precipitate of mutant PRMT1, but are absent or strongly reduced, respectively, in the precipitate of wild-type PRMT1 (Fig. 4B).


Figure 4
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  		Figure 4  Mutant PRMT1 stably interacts with nuclear substrate proteins. (A) Cells transfected with wild-type and mutant PRMT1 were disrupted by sonication. The lysate was cleared by centrifugation and then adjusted to 15 mM MgCl2 to precipitate chromatin. Precipitated material (P) was separated from the soluble supernatant (S) by centrifugation, and analyzed by staining with coomassie blue (upper panel) or western blotting to visualize PRMT1:GFP. Note the enrichment of mutant PRMT1 in the pellet fraction. (B) Total cell extracts as in (A) were immunoprecipitated with anti-GFP antibody, and the precipitate was tested by western blotting for the presence of PRMT1 (positive control), histone H3 and SAF-A.

 
Interestingly, when analyzed by confocal live cell microscopy, mutant PRMT1 displayed a granular pattern of distribution in the nucleus (Fig. 5) that was not obvious in wild-type PRMT1 expressing cells. In addition, most cells expressing mutant PRMT1 also exhibited a small number of intense dots of high enzyme concentration, very often in direct vicinity of nucleoli (inserts in Fig. 5). These dots were also observed in cells expressing wild-type PRMT1, although with much lower intensity and in fewer cells (see discussion).


Figure 5
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  		Figure 5  Comparison of the intranuclear localization of the catalytically inactive mutant of PRMT1 and wild-type PRMT1. Confocal slices of typical living cells expressing wild-type and mutant PRMT are shown. Mutant PRMT1 exhibits a more heterogeneous intranuclear distribution. In addition, small dot-like spots of enrichment, often in direct vicinity of nucleoli, are found in both wild-type and mutant PRMT1 (arrows), but appear to be more frequent in mutant PRMT1. Insert bottom right: contrast-enhanced and slightly magnified boxed region of the main image. Scale bar: 10 µm.

 
We found that expression of a catalytically inactive PRMT1 leads to a much higher degree of enrichment of the protein in the nucleus than treatment of cells with Adox (compare Herrmann et al. 2005). Additional Adox treatment of cells expressing mutant PRMT1 does only insignificantly increase further the nuclear amount of PRMT1 (Fig. 6). Apparently, inhibition by Adox and expression of a catalytically inactive enzyme both lead to nuclear accumulation via the same mechanism, the tethering of the enzyme to its substrate, and are therefore not additive in effect. We conclude that the tight association of PRMT1 with substrates such as core histones and constituents of hnRNP complexes is a direct effect of the inability of the enzyme to methylate its substrates, and that methylation is a prerequisite for the release of substrate to make it available again for nuclear export.


Figure 6
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  		Figure 6  Inhibition of methylation does not significantly affect the accumulation of catalytically inactive PRMT1 in the nucleus. Cells stably expressing mutant PRMT1 were treated for two days with periodate oxidized adenosine (Adox), a general inhibitor of cellular methylation reactions, or left untreated. Microscopic fields of typical untreated and treated cells (confocal slices) are shown, together with a quantification of the accumulation carried out as described in the legend of Fig. 3. Scale bar: 10 µm.

 
This conclusion is further corroborated by results from in vivo mobility measurements of wild-type and mutant PRMT1 by FRAP experiments. As shown in Fig. 7, treatment of cells with Adox leads to the formation of an immobile fraction in the nucleus (but not the cytoplasm) for wild-type and mutant PRMT1. The difference in mobility and immobile fraction for wild-type versus mutant is statistically insignificant, with very similar diffusion coefficients of D = 1.26 µm2/s (wild-type) and D = 1.09 µm2/s (mutant). In contrast, FRAP carried out on untreated cells reveal the presence of an 8% immobile fraction for mutant PRMT1, statistically significantly more than the 2% immobile fraction seen for wild-type PRMT1 (P < 0.05). Also, the diffusion is slower for the mutant (D = 0.88 µm2/s) than that of the wild-type enzyme (D = 1.20 µm2/s), most probably reflecting non-productive contacts of the mutant PRMT1 with potential substrates in comparison to the typical hit-and-run interaction of the active enzyme with its substrate.


Figure 7
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  		Figure 7  In vivo mobility of wild-type and mutant PRMT1. Cells expressing equal amounts of wild-type or mutant PRMT1 were used for fluorescence recovery after photobleaching (FRAP) experiments, without (left panel) or with (right panel) prior treatment with Adox to inhibit methylation. For each analysis 15–25 individual cells were examined. As shown earlier (Herrmann et al. 2005), an immobile fraction of PRMT1 is detected in the nucleus of Adox-treated cells, while cytoplasmic PRMT1 is not affected by inhibition of methylation. Note that for mutant PRMT1, there is a fraction of 8% of immobile nuclear protein even in absence of Adox (statistically significant in comparison to 2% in wild-type expressing cells), and that mutant PRMT1 also becomes more immobile by Adox-treatment (no statistically significant difference to wild-type PRMT1).

 
Interestingly, although Adox treatment of cells expressing mutant PRMT1 does not significantly increase PRMT1 nuclear localization (Fig. 6), it does appear to increase the immobile fraction mutant PRMT1 within the nucleus (Fig. 7—compare right and left panels), in a manner similar to wild-type PRMT1. In addition, mutant PRMT1 has a higher immobile fraction than wild-type PRMT1 already in non-inhibited cells, although this fraction is significantly smaller than in the case of Adox inhibition. We attribute this difference to the mode of action of PRMT1 that we have presented earlier, namely that PRMT1 firmly binds to its substrate, and releases it only after the methylation reaction has occurred (Herrmann et al. 2005). In the case of inhibition by Adox, the enzyme becomes trapped for a longer time than in the case of mutated protein, because in the latter case there are active enzyme molecules in the same oligomeric complex, which can methylate and release a bound substrate. The steady state is, therefore, shifted towards unbound enzyme which has just been released from substrate but has not yet been exported from the nucleus.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The regulation of activity and substrate interaction of protein arginine methyltransferases (PRMTs) is not yet well understood, particularly regarding their dynamics in live cells. In the present article, we show that the predominant member of the family, PRMT1, requires enzymatic activity for its shuttling between the cytoplasm and the nucleus. For the purpose of the study, we chose the splicing variant 2 of the enzyme, because we had previously shown that this variant is predominantly localized in the cytoplasm of many cell lines, but shuttles in and out of the nucleus depending on the methylation status of its substrates (Herrmann et al. 2005). Recently, the laboratory of Jocelyn Coté discovered that splicing variant 2 of PRMT1 (PRMT1v2) contains a functional nuclear export signal in its alternative amino-terminus that is lacking in other splice isoforms of PRMT1 (Goulet et al. 2007). The authors also provided evidence that PRMT1v2 might be involved in breast cancer due to mis-regulated protein methylation. Therefore, PRMT1v2 appears to be an excellent candidate to study mechanisms that contribute to regulation of members of the PRMT family of enzymes and might provide a link to pathophysiological consequences.

We introduced a three-residue mutation into the S-adenosyl methionine binding site of PRMT1v2 to create a catalytically inactive construct. This mutation is particularly suitable for the purpose of this study, as the inactivation is the result of failure of the mutant to bind the methyl donor, whereas protein domains responsible for substrate recognition of the enzyme, its active center, or the interface necessary for homo-oligomerization remain unaffected. Thus, this mutant should faithfully mimic the active enzyme with regard to substrate recognition and interaction with endogenous PRMT1. We found that the catalytically inactive PRMT1 is properly incorporated into complexes with endogenous PRMT1. However, in comparison to wild-type PRMT1 that was investigated in parallel, inactive PRMT1 was expressed in significantly lower amounts after selection for stably expressing cells. While mutant PRMT1 was already expressed in slightly decreased amounts (Fig. 1A) directly after transfection, the expressed protein levels became different by a factor of 10 as selection proceeded. In addition, we noticed a slight increase of dead cells in the first days after transfection of mutant PRMT1. The decrease in expression was specific for the PRMT1 mutant, as we did never observe a similar effect during selection of other GFP fusion proteins expressed from the same vector/promoter, including seven other members of the PRMT family (to be published elsewhere), hnRNP-C, SAF-A and Actin. We conclude that the expression of higher amounts of mutant PRMT1 leads to a growth disadvantage during the time of selection. The most probable reason is that the mutant, by forming hetero-oligomers with endogenous PRMT1, decreases the specific activity of the complex depending on the ratio of mutant to endogenous PRMT1. This is compatible with earlier findings that the VLD to AAA mutation acts dominantly negative when tested in vitro (Wada et al. 2002) for PRMT1, and also in vivo for CARM1/PRMT4 (Lee et al. 2006). Thus, only cells expressing tolerable amounts of mutant PRMT1 will survive the selection. Wild-type PRMT1, which is also incorporated into complexes with the endogenous enzyme, does not affect the specific activity of the complex, and therefore does not exert a negative effect.

Confocal live cell microscopy revealed that the catalytically inactive mutant of PRMT1v2 strongly accumulated in the nucleus, whereas the wild-type PRMT1v2 was predominantly cytoplasmic, in line with our original observations (Herrmann et al. 2005) and the study of Goulet et al. (2007). The accumulation was qualitatively similar but much more pronounced than the accumulation of wild-type GFP-PRMT1 under conditions of inhibition of methylation by oxidized adenosine (Herrmann et al. 2005). When cells expressing inactive PRMT1 were treated with Adox, the accumulation was not further increased. This is compatible with the finding that PRMT1 tightly associates with natural substrates such as histones and members of the hnRNP family of proteins (Herrmann et al. 2004) until the enzyme has finished the methylation reaction. Interestingly, microscopic analysis also showed that mutant PRMT1 localizes to a small number of intranuclear dots in most cells, which were only rarely seen in cells expressing wild-type PRMT1. Based on the number of dots per nucleus, their shape and their localization close to nucleoli, these dots may be Cajal bodies, in agreement with findings of Yanagida et al., who showed that PRMT1 is enriched in Cajal bodies because of its interaction with fibrillarin, and may be involved in snRNP biogenesis and pre-rRNA processing (Jady et al. 2003; Yanagida et al. 2004).

Our results show that the catalytically inactive mutant of PRMT1v2 is highly enriched in the nucleus, despite its intact nuclear export signal. Also, mutant PRMT1 is associated with histones and SAF-A, as determined in co-immunoprecipitation experiments. These results fully confirm identical experiments done before with Adox inhibition of PRMT1 (Herrmann et al. 2005), and effectively rule out the alternative possibility that nuclear enrichment of mutant PRMT1 is due to interference in nuclear export processes. We conclude that it is the tight association of PRMT1 with substrates such as core histones, constituents of hnRNP particles and fibrillarin that tethers the inactive enzyme to nuclear substructures so that it is not available for export. These data provide a firm and quantitative basis for the role of protein shuttling in the reaction cycle of PRMT1v2. It is interesting to note that other splicing variants of PRMT1 appear to be evenly distributed between cytoplasm and nucleus, or are more abundant in the nucleus, although none of them has a nuclear localization signal (Goulet et al. 2007); how they enter the nucleus is currently not completely clear. It is conceivable that they are imported into the nucleus piggybacked on unmethylated substrates (also see Herrmann et al. 2005), where they carry out the methylation reaction and are released from the substrate. While other variants appear to remain nuclear, at least for some time, PRMT1v2 is exported from the nucleus owing to its nuclear export signal. This shuttling cycle might ensure that PRMT1v2 leaves the nucleus and is sequestered away from nuclear substrates unless it is co-imported with a newly expressed, unmethylated substrate. Interestingly, even though PRMT1 also has cytoplasmic substrates (Le Romancer et al. 2008), we have not found evidence for stable binding of the enzyme to substrates in the cytoplasm. We do currently not know why nuclear localization of PRMT1v2 is regulated in a tighter way than that of other splicing isoforms, which are found in both the cytoplasm and the nucleus, and lack a nuclear export signal (Goulet et al. 2007). One reason may be that splicing isoforms of PRMT1 differ in substrate specificity, and that tighter regulation of PRMT1v2 substrate methylation is essential for cell survival, growth or some other vital cellular function. This might also explain the potential involvement of PRMT1v2 in breast cancer development discussed by Goulet et al. (2007). Clearly, more work is necessary and on the way to better understand the determinants of substrate recognition of different splicing isoforms of PRMT1.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture, transfection, and treatment with periodate-oxidized adenosine

Human embryonic kidney cells (HEK293) were cultivated on plastic dishes in DMEM with 10% fetal calf serum in a humidified atmosphere containing 5% CO2, and were split 1 : 5 every second day. Cells were transfected by polyethylenimine (Boussif et al. 1995) with expression vectors encoding EGFP fusions of wild-type or mutant PRMT1v2, respectively, and stably expressing cell lines were created by selection with G418 for 4 weeks. After selection, cells were analyzed and sorted for GFP fluorescence with a FACSAria cell sorter (BD Biosciences).

For inhibition of methylation or for preparation of hypomethylated cell extracts, the medium of HEK293 cells was supplemented with 15 µM of periodate-oxidized adenosine (Adenosine-2'-3'-dialdehyde, Sigma A7154), and cells were cultured for additional 48 h before they were analyzed or harvested.

In vitro mutagenesis of PRMT1v2

The catalytically inactive mutant of PRMT1 was created by in vitro mutagenesis following the protocol of the Stratagene Quikchange mutagenesis kit, using primers 5'-ACC TCT TCA AGG ACA AGG TGG CGG CGG CCG TCG GCT CGG GCA CCG GCA T-3' and 5'-ATG CCG GTG CCC GAG CCG ACG GCC GCC GCC ACC TTG TCC TTG AAG AGG T-3'. These primers introduce a three-residue mutation, changing residues V68, L69 and D70 in the S-Adenosyl-Methionine binding site of PRMT1v2 to alanines. The resulting clones were sequence-verified and transfected as described above to obtain stably expressing cell lines.

Live cell microscopy and FRAP experiments

For live cell microscopy, cells were split onto 35 mm culture dishes with a glass bottom (Mattek), and analyzed 2 days after splitting either untreated or treated with Adox as described above. For the analysis, cells were placed on the heated stage of a Zeiss Meta 510 confocal laser-scanning microscope. FRAP experiments were carried out as described previously (Herrmann et al. 2005; Mearini & Fackelmayer 2006). For quantitative analysis of protein abundance in the nucleus or the cytoplasm, a minimum of 100 individual cells were measured, and mean pixel intensities in specified regions of interest (ROI) were determined with the freeware image analysis software ImageJ by Wayne Rasband (http://rsb.info.nih.gov/ij/).

In vitro methylation assays were carried out essentially as described previously (Herrmann et al. 2004). As a substrate, we used hypomethylated extract from HEK293 cells treated with 15 µM of periodate-oxidized adenosin (Adenosin-2'-3'-dialdehyde, Sigma A7154) for 48 h. The extract was pretreated at 70 °C for 10 min to inactivate endogenous methyltransferases, and centrifuged for 10 min at full speed. The supernatant was adjusted to 1 x PBS (final concentrations: 1.5 mM KH2PO4, 12.7 mM K2HPO4, 138 mM NaCl, 2.7 mM KCl, pH 7.5) by adding concentrated PBS stock solution, and centrifuged again. Per reaction, 20 µL of extract was combined with PRMT1 immunoprecipitated from stably or transiently transfected cells, 5 µCi S-adenosyl-L-[methyl-3H]methionine (Amersham Biosciences TRK865), and reactions were incubated for 2 h at 37 °C. The reaction mixture was then resolved by SDS-page and radioactively labeled proteins were visualized by fluorography with enHance reagent (NEN Dupont).

Immunoprecipitation and Western blotting assays were carried out exactly as described previously (Herrmann et al. 2004). In several cases, RIPA buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% Nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS) was used to increase the stringency of immunoprecipitation and attempt to break the interaction between transfected PRMT1 and endogenous PRMT1. For western blotting, we used antibodies A33 and F339 (both from Cell Signalling) and 07-404 (Upstate) against PRMT1, ab1791 (abcam) against histone H3, and anti-GFP (Roche).


   Acknowledgements
 
Authors thank Arne Düsedau from the Heinrich-Pette-Institute, Hamburg, Germany, for expert help with cell sorting, and previous and current members of the lab, especially Peter Pably and Carmen Eckerich, for discussions and critically reading the manuscript. This work was supported by grant Fa376/3–1 of the Deutsche Forschungsgemeinschaft (DFG) to F.O.F.


   Footnotes
 
Communicated by: Robert Roeder

* Correspondence: frank{at}fackelmayer.de


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bedford, M.T. & Richard, S. (2005) Arginine methylation an emerging regulator of protein function. Mol. Cell 18, 263–272.[CrossRef][Medline]

Boisvert, F.M., Cote, J., Boulanger, M.C. & Richard, S. (2003) A proteomic analysis of arginine-methylated protein complexes. Mol. Cell. Proteomics 2, 1319–1330.[Abstract/Free Full Text]

Boussif, O., Lezoualc’h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B. & Behr, J.P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA 92, 7297–7301.[Abstract/Free Full Text]

Cook, J.R., Lee, J.H., Yang, Z.H., Krause, C.D., Herth, N., Hoffmann, R. & Pestka, S. (2006) FBXO11/PRMT9, a new protein arginine methyltransferase, symmetrically dimethylates arginine residues. Biochem. Biophys. Res. Commun. 342, 472–481.[CrossRef][Medline]

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Received: 26 June 2008
Accepted: 21 November 2008




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F. Herrmann, P. Pably, C. Eckerich, M. T. Bedford, and F. O. Fackelmayer
Human protein arginine methyltransferases in vivo - distinct properties of eight canonical members of the PRMT family
J. Cell Sci., March 1, 2009; 122(5): 667 - 677.
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