The p160 family of coactivators, SRC-1, GRIP1/TIF2, and p/CIP, mediate transcriptional activation by nuclear hormone receptors. Coactivator-associated arginine methyltransferase 1 (CARM1), a previously unidentified protein that binds to the carboxyl-terminal region of p160 coactivators, enhanced transcriptional activation by nuclear receptors, but only when GRIP1 or SRC-1a was coexpressed. Thus, CARM1 functions as a secondary coactivator through its association with p160 coactivators. CARM1 can methylate histone H3 in vitro, and a mutation in the putative S-adenosylmethionine binding domain of CARM1 substantially reduced both methyltransferase and coactivator activities. Thus, coactivator-mediated methylation of proteins in the transcription machinery may contribute to transcriptional regulation.

Transcriptional induction by interferons requires the tyrosine and serine phosphorylation of STAT transcription factors. The N-terminal region is highly homologous among the STAT proteins and surrounds a completely conserved arginine residue. Here we demonstrate arginine methylation of STAT1 by the protein arginine methyl-transferase PRMT1 as a novel requirement for IFNalpha/beta-induced transcription. Methyl-thioadenosine, a methyl-transferase inhibitor that accumulates in many transformed cells, inhibits STAT1-mediated IFN responses. This inhibition arises from impaired STAT1-DNA binding due to an increased association of the STAT inhibitor PIAS1 with phosphorylated STAT1 dimers in the absence of arginine methylation. Thus, arginine methylation of STAT1 is an additional posttranslational modification regulating transcription factor function, and alteration of arginine methylation might be responsible for the lack of interferon responsiveness observed in many malignancies.

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Posttranslational modification of proteins within T cell receptor signaling cascades allows T lymphocytes to rapidly initiate an appropriate immune response. Here we report a role for arginine methylation in regulating cytokine gene transcription in the T helper lymphocyte. Inhibition of arginine methylation impaired the expression of several cytokine genes, including the signature type 1 and type 2 helper cytokines, interferon gamma, and interleukin-4. T cell receptor signaling increased expression of the protein arginine methyltransferase PRMT1, which in turn methylated the nuclear factor of activated T cells (NFAT) cofactor protein, NIP45. Arginine methylation of the amino terminus of NIP45 modulated its interaction with NFAT and resulted in augmented cytokine production, while T cells from mice lacking NIP45 had impaired expression of IFNgamma and IL-4. Covalent modification of NIP45 by arginine methylation is an important mechanism of regulating the expression of NFAT-dependent cytokine genes.

RNA helicase A (RHA) undergoes nuclear translocation via a classical import mechanism utilizing karyopherin beta. The nuclear transport domain (NTD) of RHA is known to be necessary and sufficient for its bi-directional nuclear trafficking. We report here that arginine methylation is a novel requirement for NTDmediated nuclear import. Nuclear translocation of glutathione S-transferase (GST)-NTD fusion proteins is abrogated by arginine-methylation inhibitors. However, in vitro arginine-methylation of GST-NTD prior to injection allows the fusion protein to localize to the nucleus in the presence of methylation inhibitors. Removal of the arginine-rich C-terminal region negates the effects of the methylation inhibitors on NTD import, suggesting that methylation of the NTD C terminus the relieves the cytoplasmic retention of RHA. The NTD physically interacts with PRMT1, the major protein arginine methyltransferase. These findings provide evidence for a novel arginine methylation-dependent regulatory pathway controlling the nuclear import of RHA.Arginine di-methylation, which is restricted to eukaryotic cells, occurs frequently in the context of RGG tripeptides (1, 2). Two classes of protein arginine methyltransferases (PRMT) 1 have been characterized and classified based on the symmetry of their reaction products. Type I PRMTs account for the formation of asymmetric N G ,N G -dimethylarginine, whereas type II enzymes catalyze the formation of symmetric N G ,N G Ј-dimethylarginine. Five related enzymes that catalyze asymmetric arginine methylation have thus far been identified. The majority of Type I PRMT activity in eukaryotic cells appears to be accounted for by PRMT1 and its functional yeast homologue Hmt1/Rmt1 (3). Several recent studies have illustrated an important function of arginine methylation in the regulation of protein function. Methylation of Sam68, a proline-rich srckinase substrate known to interact with WW or Src homology 3 domain containing signaling proteins, decreases its affinity for Src homology 3 domains but does not alter binding to WW motif-containing proteins (4). Similarly, arginine methylation of the STAT1 (signal transducer and activator of transcription) transcription factor decreases its affinity for its inhibitor PIAS1 (protein inhibitor of activated STAT 1), thereby modulating interferon-induced gene transcription (5). A positive modulatory effect of arginine methylation on protein-protein interaction is observed with the Type II PRMT substrates SmD1 and SmD3, which require methylation for efficient binding to the spinal muscular atrophy gene product, SMN (6,7).Proteins within eukaryotic cells that shuttle across the nuclear envelope to facilitate proper cell function undergo nucleocytoplasmic transport by utilizing specific nuclear transport receptors, which ferry the cargo proteins through the nuclear pore complex (NPC). A signal sequence domain contained within the cargo proteins allows for the recognition of the protein by the transport receptor (8). The classical and by far most we...