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. 2013;3:1311.
doi: 10.1038/srep01311.

Loss of the Major Type I Arginine Methyltransferase PRMT1 Causes Substrate Scavenging by Other PRMTs

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Free PMC article

Loss of the Major Type I Arginine Methyltransferase PRMT1 Causes Substrate Scavenging by Other PRMTs

Surbhi Dhar et al. Sci Rep. .
Free PMC article

Abstract

Arginine methylation is a common posttranslational modification that is found on both histone and non-histone proteins. Three types of arginine methylation exist in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). PRMT1 is the primary methyltransferase that deposits the ADMA mark, and it accounts for over 90% of this type of methylation. Here, we show that with the loss of PRMT1 activity, there are major increases in global MMA and SDMA levels, as detected by type-specific antibodies. Amino acid analysis confirms that MMA and SDMA levels accumulate when ADMA levels are reduced. These findings reveal the dynamic interplay between different arginine methylation types in the cells, and that the pre-existence of the dominant ADMA mark can block the occurrence of SDMA and MMA marks on the same substrate. This study provides clear evidence of competition for different arginine methylation types on the same substrates.

Conflict of interest statement

MTB is a cofounder of EpiCypher. MJC and AG work at Cell Signaling Technology Inc.

Figures

Figure 1
Figure 1. Characterization of the monomethylarginine antibodies.
(a) PRMT1fl/− ER-Cre MEFs were untreated or treated with 4-hydroxytamoxifen (OHT) for 8 days. Whole cell lysates were prepared and immunoblotted with monomethylarginine (MMA1-5) and asymmetric dimethylarginine (ADMA) antibodies. MMA antibodies are highly immunoreactive towards PRMT1 knockout (−/−) MEFs compared to the wild-type (+/+) counterparts suggesting that the KO cells have a rich pool of mono-methylated proteins. ADMA antibody showed reduced immunoreactivity in PRMT1 −/− cells, which suggests that they have lower levels of dimethylated proteins. Whole cell extracts of PRMT3 (b), CARM1 (c) and PRMT6 (d) +/+ and −/− MEFs were prepared and immunoblotted with MMA1-5 antibodies. The immunoreactivity patterns are the same in +/+ versus −/− cells. PRMT3 (b), PRMT5 (e) and PRMT6 (d) MEFs were also blotted with ADMA antibody, revealing no changes in banding patterns. CARM1 (c) MEFs were immunoblotted with H3R17 antibody (H3R17me2a, Millipore). Although this antibody was originally generated to recognize dimethyl-Arg17 on histone H3, it was shown to behave as a pan-antibody. (c) It recognized a number of proteins in +/+ cells that are absent in CARM1 −/− cells, suggesting a decrease of dimethylation in these cells. (e) PRMT5 control and knockdown (KD) HeLa cells were immunoblotted with MMA1-5 and symmetric dimethylarginine (SDMA) antibodies. MMA antibodies do not show significant differences in band patterns in control versus KD cells except for a doublet of bands, which appeared at 25 kDa in KD cells (indicated with solid white arrows). SDMA antibody showed reduced immunoreactivity in KD cells. Western analyses with αPRMT1 (a), αPRMT3 (b), αCARM1 (c), αPRMT5 (e) and αPRMT6 (marked with asterisk) (d) antibodies show the loss of these PRMTs in the respective −/− cell lines. All lysates were blotted with β-actin antibody to visualize equivalent loading.
Figure 2
Figure 2. Arginine methylation trends in inducible PRMT1-knockout MEFs.
PRMT1fl/− ER-Cre MEFs were untreated or treated with 4-hydroxytamoxifen (OHT) for 10 days. Total cell lysates from different days of treatment were immunoblotted with antibodies against monomethylarginine (MMA2) (a), asymmetric dimethylarginine (ADMA) (b), symmetric dimethylarginine (SDMA), PRMT1 and β-actin (c).
Figure 3
Figure 3. Quantification of MMA, ADMA, SDMA, and arginine levels in protein hydrolysates of wild-type and PRMT1-knockout MEFs.
Cell pellets from PRMT1 wild-type and knockout MEFs were acid hydrolyzed and the resulting amino acids separated by high-resolution cation exchange chromatography as described in "Methods". The separation of standards (1 μmol ) of ADMA, SDMA, and MMA/arginine with ninhydrin detection as described by Zurita-Lopez et al. (2012) is shown in the control chromatograph (a). The separation of these amino acids is typical, although small changes in the elution times can occur between runs. Cell hydrolysates were then chromatographed without standard amino acids and fractions analyzed by reverse-phase HPLC after derivatization with OPA for fluorescence quantification as described in "Methods". HPLC conditions were optimized to separate the large pool of arginine from ADMA and SDMA in wild-type (b) and PRMT1 knockout (c) and from MMA in wild-type (d) and PRMT1 knockout (e) samples (Supplemental Fig. 5). The total amount of a given species was quantified by summing the integrated area under the curve for all HPLC fractions containing the respective species that are consistent with the migration on the cation-exchange column.

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