Arginine Methylation of RNA Helicase A Determines its Subcellular Localization

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 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. GST antisera FITC-conjugated anti-mouse


INTRODUCTION
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) 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 -dimethyl-arginine, whereas type II enzymes catalyze the formation of symmetric N G ,N' G -dimethyl-arginine. 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 src-kinase substrate known to interact with WW-or SH3-domain containing signaling proteins, decreases its affinity for SH3-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 in order to facilitate proper cell function undergo nucleo-cytoplasmic 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 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 α/β heterodimer as the import receptor, is both necessary and sufficient for protein transport across the NPC. During the import process, importin α binds the NLS-containing protein and serves as a link to importin β, which in turn interacts with the NPC. Recently, several proteins containing arginine-rich motifs within their NLS have been discovered that can bind to importin β independently of importin α (10,11).
RNA Helicase A (RHA), whose nuclear import was previously thought to employ importin α and β, contains several arginine residues within its nuclear localization signal sequence. Mammalian RHA, also known as nuclear DNA helicase II, was first identified as a protein capable of unwinding double stranded RNA as well as double stranded DNA sequences, and has since been shown to fulfill multiple roles in mammalian cells (12)(13)(14). As such, RHA has been implicated in transcription by binding to promoter-proximal sequences (15), bridging to the CBP/p300 complex (16) and interacting with RNA Polymerase II (17). An additional function of RHA, which has similarity to yeast pre-mRNA splicing factors prp2, prp16 and prp22 (12), appears to be in cellular RNA splicing and processing through binding to the hnRNP proteins K, C1, and A as well as snRNP protein (14).
RHA has also been reported to increase retroviral RNA transcription by binding to the HIV trans-activation response elements (TAR) (18,19).
Additionally, RHA promotes the export of mRNA transcripts through binding to TAP and HAP95 (20,21), and plays a role in the export of CTE-containing viral transcripts (22,23). Because of these multiple roles of RHA in the cell, it is important to elucidate the mechanism which governs the nucleo-cytoplasmic distribution of RHA.
The C-terminus of RHA contains a 110 amino acid bi-directional nuclear transport domain (NTD) that is necessary and sufficient for both nuclear import and export (24). Heterokaryon 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.
To date, phosphorylation is the only signal transduction mechanism shown to be involved in nuclear import. Since the NTD of RHA contains several RG/RGG motifs, we decided to investigate whether these residues are methylated by PRMT1, and whether the nuclear translocation of RHA is altered depending on its methylation state. Indeed we find that the NTD of RHA can be methylated by by guest on March 24, 2020 http://www.jbc.org/ Downloaded from PRMT1 in-vitro, and that this methylation is necessary for NTD nuclear translocation. As such, our findings provide the first direct evidence that arginine methylation within a mammalian NLS regulates the function of the nuclear import signal.

Results and Discussion
RHA contains a 110 amino acid (pos. 1150-1259) bi-directional nuclear transport domain (NTD) that is necessary and sufficient for both nuclear import and export of the enzyme. This region harbors an NLS as well as a nuclear export sequence (NES). Mutation or deletion of Lys 1162 causes the loss of RHA nuclear import, whereas deletion of aa 1221-1259 impairs the nuclear export of the protein (Fig. 1A). The "RGG" motifs found in the arginine-rich sequences of the NTD follow the consensus substrate recognition site for PRMTs (Fig. 1B). As PRMT1 accounts for the vast majority of PRMT activity in the cell, we decided to test whether the NTD can indeed function as a substrate for this enzyme. As shown in Fig. 2, PRMT1 is able to methylate GST fusion proteins of the full length NTD (lane 1) as well as of both the ΔK62 and 508 deletion mutants (lanes 2 and 3, respectively), whereas GST alone was not methylated under these conditions (lane 4).
We next wanted to determine whether the NTD can interact with endogenous PRMT1 in vivo. Using the GST-NTD fusion proteins as bait, we performed pulldown experiments in U266 whole cell lysates. As expected, PRMT1 associated with the GST-fusion proteins of the full-length NTD as well as the ΔK62 deletion mutant, but not with GST alone. Intriguingly, PRMT1 also did not bind to the GST-508 fusion protein, even though this protein is methylated by PRMT1 in vitro ( Fig. 2B lanes 3, 4, and 6). It thus appears that the C-terminal domain of the NTD, which harbors multiple RG/RGG motifs that are absent in the 508 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from truncation, may be crucial for the high-affinity NTD/PRMT1 interaction required to sustain the association under the experimental conditions. In order to confirm the association of PRMT1 with RHA, we also performed pull-down experiments with GST-PRMT1 to test for its ability to associate with full-length endogenous RHA (Fig. 2C). Indeed, RHA bound to GST-PRMT1, but not to GST alone, supporting the concept of a physical interaction between these two proteins.
The above experiments established that PRMT1 associates with RHA and is able to methylate full-length NTD as well as the ΔK62 and 508 deletion mutants in vitro. The localization of the RGG motifs suggested that methylation of the arginine within the NTD might exert a regulatory effect on the subcellular localization of RHA. To test this hypothesis, we performed microinjections of the full length GST-NTD as well as the GST-ΔK62 and GST-508 deletion mutants, followed by immunofluorescence analysis with anti-GST antibodies. Previous studies had demonstrated that the GST-NTD fusion protein displays a nuclear steady state localization despite its shuttling between cytoplasm and nucleus (24). Accordingly, when full-length GST-NTD was injected into the cytoplasm of HeLa cells, it appeared in the nucleus in 100% of the injected cells (Fig 3A, (25). When these methylation inhibitors were added to cells prior to microinjection, more than 85% of the injected cells displayed a clear inhibition of nuclear import of the GST-NTD (Fig.   3A, bottom row). Importantly, when the truncated GST-508 fusion protein which lacks several of the RGG motifs was injected, it localized to the nucleus even in the presence of the methylation inhibitors (Fig. 3B). Together, these findings not only demonstrate that the RGG-motifs in the C-terminal of the NTD, which are absent in GST-508, are not necessary for the nuclear localization, but also argue the arginine methylation of its C-terminal region overcomes a negative effect exerted by this region on the nuclear import of the GST-NTD.
One concern with the interpretation of the above experiments is the possibility that the abrogation of GST-NTD nuclear import by the PRMT inhibitors might not be due to blocking of the methylation of the NTD itself. Even though the fact that the nuclear translocation of the GST-508 is unaffected by the methylation inhibitor argues against this possibility, we could not exclude that the methylation inhibitors do not only target the methylation of the NTD, but also that of additional cofactor(s) that might be required for its nuclear translocation. We therefore decided to subject the GST-NTD to in-vitro arginine methylation by recombinant PRMT1 prior to microinjection of such arginine methylated fusion protein into untreated cells, or into cells that had previously been exposed to the methylation by guest on March 24, 2020 http://www.jbc.org/ Downloaded from inhibitors. As shown in Fig. 3A, unmethylated GST-NTD is restricted to the cytoplasm when injected into cells treated with the methylation inhibitors. In contrast, when in-vitro arginine methylated GST-NTD is introduced into these cells, we found that the pre-methylated GST-NTD was still able to localize to the nucleus (Fig. 3C). These results clearly establish that the arginine methylation of the NTD itself is a crucial requirement for the nuclear import of the NTD fusion protein. The above results and those of additional nuclear and cytoplasmic microinjections are summarized in Table 1. To exclude the possibility that our observations were an artifact of the isolated NTD, we performed heterokaryon assays with GFP-RHA(ApaI) as previously described (24). Exposure of the cells to methylation inhibitors prevented the movement between the nuclei as anticipated (data not shown).
The role of phosphorylation in the control of nuclear import in mammalian cells has been well documented, in contrast, a function for arginine methylation as a requirement for this process has not been previously reported. Precedence exists for a role of arginine methylation in cytoplasmic-nuclear transport in S.
cerevisiae. There, the RGG-rich domain of Npl 3p, when methylated by Hmt1/Rmt1, inhibits the phosphorylation of its NLS and its nuclear import. In a converse manner, arginine methylation is known to regulate the nuclear export of hnRNPs in yeast.
Our results presented here provide the first evidence of arginine methylationdependent nuclear import in mammalian cells. Consistent with a role for arginine methylation in NTD-mediated nuclear import, we find that the RHA-NTD can be methylated in vitro by PRMT1. Moreover, the NTD interacts with PRMT1 in GST pull-down assays, and conversely, GST-PRMT1 is able to bind endogenous RHA. Nuclear import mediated by the NTD of RHA is abrogated in the presence of inhibitors of PRMTs. This block of nuclear translocation can be overcome by in-vitro methylating of the NTD prior to microinjection. Our studies summarized in Table I, however, do not provide any evidence for a role of arginine methylation in NTD-mediated nuclear export. Intriguingly, GST-508, created by removal of the RGG-rich C-terminus of the NTD, is able to enter the nucleus even in the presence of inhibitors of arginine methylation.
One potential explanation for the differential dependence of RHA-NTD and RHA-508 on arginine methylation for their nuclear localization might be the presence of an unidentified cytoplasmic "retention factor" which binds to the unmethylated RGG-rich domain of the NTD and thereby prevents access of importin β to the NTD-NLS. Arginine methylation of the NTD causes the inhibitory protein to be released from RHA and thus allows for the exposure of the NLS, a process which is abrogated by inhibition of the methylation reaction. Removal of the RGG-rich C-terminal domain of the NTD eliminates the docking site for this putative inhibitor protein and allows nuclear import to proceed even in the presence of the PRMT inhibitors. This model is consistent with the finding that PRMT1 binds to GST-NTD, but not to GST-508. In summary, we have shown that arginine methylation affects NTD-mediated nuclear import via an RGG-rich NLS, thus, it is likely that methylation affects the import of other proteins containing arginine-rich nuclear localization signals as well. As such, methylation may prove to be an important posttranslational modification of proteins requiring nuclear import/export for proper cellular function.