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First published online 10 February 2009
doi: 10.1242/jcs.039933

Journal of Cell Science 122, 667-677 (2009)
Published by The Company of Biologists 2009
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Human protein arginine methyltransferases in vivo – distinct properties of eight canonical members of the PRMT family

Frank Herrmann1, Peter Pably2, Carmen Eckerich2, Mark T. Bedford3 and Frank O. Fackelmayer2,*

1 EMBL-CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), c/Dr. Aiguader 88, 08003 Barcelona, Spain
2 Biomedical Research Institute, Foundation for Research and Technology Hellas, 45110 Ioannina, Greece
3 Department of Carcinogenesis, M. D. Anderson Cancer Center, University of Texas, 1808 Park Road 1C, Smithville, TX 78957, USA


Figure 1
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  		Fig. 1. Expression and activity of human PRMT1-PRMT8 fused to EGFP. (A) Schematic representation of the eight canonical members of the human PRMT family. The highly conserved PRMT core region (gray), signature motifs I, post I, II, and III (black) and the conserved THW loop (red), present in all PRMTs, are indicated. Note that PRMT7 has a duplication of these motifs and PRMT2 and PRMT3 have an N-terminal SH3 domain (orange) or a zinc-finger domain (green), respectively. The nuclear localization signal (NLS, blue) targets PRMT6 to the nucleus, whereas N-terminal myristoylation (~) tethers PRMT8 to the plasma membrane. The size of individual PRMTs is indicated at each C-terminus. (B) Human embryonic kidney (HEK293) cells were stably transfected with expression vectors encoding PRMT1-GFP to PRMT8-GFP. Total cell extracts were separated by SDS-PAGE, blotted to a polyvinylidene difluoride membrane, and probed with antibodies specific for GFP. Equal amounts of total cell extract were used in each lane, verified by using hnRNP-C as a loading control. (C) Methylation activity assays. Individual PRMTs were immunoprecipitated from the total cell extracts described in B, by using anti-GFP antibodies. Activity assays were carried out with hypomethylated extracts (upper panel) or recombinant core histones (lower panel) as described in the Materials and Methods.

 

Figure 2
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  		Fig. 2. Determination of complex sizes of PRMT1-PRMT7 by glycerol gradient centrifugation. A mixture of total cell extracts from cells stably expressing PRMT1-GFP to PRMT7-GFP fusion proteins was loaded on a 10-30% glycerol gradient and proteins were separated by centrifugation for 20 hours at 30,000 r.p.m. The gradient was fractionated from the top, and aliquots of individual fractions were resolved by SDS-PAGE. Proteins were detected by western blotting using anti-GFP antibodies for detection (top panel). PRMT-GFP fusion proteins are denoted by their respective numbers (1-7). The sedimentation of endogenous PRMTs was investigated similarly, using extracts from nontransfected cells and commercially available antibodies for PRMT1 to PRMT7. BSA (66 kDa, 4.2S), β-amylase (200 kDa, 8.9S) and apoferritin (443 kDa, 17.6S) were used as sedimentation markers in an identical gradient run in parallel. Note that the expressed GFP fusion proteins of all PRMTs except PRMT5 sediment similarly to their endogenous counterparts.

 

Figure 3
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  		Fig. 3. Subcellular distribution of PRMTs in HEK293 cells. Confocal slices through typical cells that stably express PRMT1-GFP to PRMT8-GFP fusion proteins. For reference, the same cells were analyzed by differential interference contrast (DIC). Scale bar: 25 µm.

 

Figure 4
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  		Fig. 4. Subcellular distribution of PRMT1 and PRMT4 show cell-type specificity. Cell lines stably expressing PRMT1-GFP or PRMT4-GFP were created from HEK293 (human embronic kidney), U2OS (human osteosarcoma), HeLa (human cervix carcinoma) and MCF-7 (human breast adenocarcinoma) cells. Fields of typical cells for each construct and cell line are shown. Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. Photobleaching experiments reveal in vivo dynamics of PRMTs. Selected images from confocal FRAP experiments on HEK293 cells that stably express PRMT1-GFP to PRMT8-GFP. Typical cells are shown before and directly after photobleaching, and at distinct time points during fluorescence recovery. Bleaching was performed using a rectangular region spanning both the cytoplasm and the nucleus.

 

Figure 6
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  		Fig. 6. Quantitative analysis of FRAP experiments. Mobility of PRMTs was quantified individually for the nuclear (A) and the cytoplasmic (B) fraction of the proteins with or without oxidized adenosine (Adox) treatment. Note that PRMT5 lacks a nuclear fraction, and PRMT6 lacks a cytoplasmic fraction. The recovery curve of membrane bound PRMT8 is shown separately (C). Each recovery curve represents the mean of 12-22 individual cells.

 

Figure 7
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  		Fig. 7. Diffusion characteristics of PRMT complexes investigated by fluorescence correlation spectroscopy. Comparison of normalized auto correlation functions of diluted total cell extracts from cells stably expressing GFP fusion proteins. Cells were treated with oxidized adenosine (Adox) (B) 2 days prior to harvesting, or left untreated (A). Note that PRMT1, PRMT4 and PRMT5 show diffusion characteristics of high molecular weight complexes. No significant difference between auto correlation functions in –Adox and +Adox samples was observed.

 

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© The Company of Biologists Ltd 2009