Abstract
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 NTD-mediated 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.
Highlights
We report here that receptors, which ferry the cargo proteins through the nuclear arginine methylation is a novel requirement for nuclear transport domain (NTD)- pore complex (NPC)
Nuclear translocation of glu- within the cargo proteins allows for the recognition of the tathione S-transferase (GST)-NTD fusion proteins is abrogated by arginine-methylation inhibitors
We find that the NTD of RNA helicase A (RHA) can be methylated by PRMT1 in vitro and that this methylation is necessary for NTD nuclear translocation
Summary
Kinase substrate known to interact with WW or Src homology 3 domain containing signaling proteins, decreases its affinity for Received for publication, November 24, 2003, and in revised form, April 7, 2004. 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. The classical and by far most well characterized nuclear localization sequence (NLS), which consists of multiple basic amino acids including lysine and arginine residues, was originally identified in the SV40 large T antigen [9] This NLS, which utilizes an importin ␣/ heterodimer as the import receptor, is both necessary and sufficient for protein transport across the NPC. Arginine Methylation of RNA Helicase A karyon analysis demonstrated that the RG/RGG-rich NTD confers bi-directional shuttling activity when linked to GST or other non-transport proteins, even though immunofluorescence studies revealed that the NTD confers a primarily nuclear steady-state localization onto the fusion protein. Our findings provide the first direct evidence that arginine methylation within a mammalian NLS regulates the function of the nuclear import signal
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