The coordinated and sequential events that lead to regulated gene expression in eukaryotes are extremely complex, spanning across cellular compartments and requiring numerous protein and RNA factors at various stages and at specific locations. When a single protein factor, such as SF2/ASF, is involved in multiple posttranscriptional events, it is essential that its movements between cellular compartments be tightly regulated. SF2/ASF is a shuttling protein that shows predominant nuclear localization in the steady state (13
). Within the nucleus, SF2/ASF accumulates in nuclear speckles, and its recruitment to active sites of transcription is modulated by the phosphorylation of Ser residues in the RS domain and various protein-protein interactions (46
). The phosphorylation of SF2/ASF also modulates its subcellular localization (32
). The RS domain is required for the shuttling of SF2/ASF (13
) and contributes to its nuclear localization (12
Here we have demonstrated that additional signals that control the cellular localization of SF2/ASF are present in the linker between RRM1 and RRM2. The Arg residues in this linker region, in particular R93, R97, and R109, are methylated (48
) and are important for correct localization, as we found that mutating these residues simultaneously to Ala resulted in cytoplasmic rather than nuclear accumulation.
The role of the SF2/ASF inter-RRM linker in RNA binding is unclear, as a structure of full-length SF2/ASF or its two RRMs in complex with RNA is lacking. Evidence from the structures of other two-RRM proteins, such as UP1, Sex-lethal, and nucleolin, indicates that the inter-RRM linkers, which are disordered when unbound, cooperate with the RRMs in binding the nucleic acid by providing increased affinity and specificity (42
). In our study, the triple-Lys mutant was functionally indistinguishable from wt-SF2/ASF, and the triple-Ala mutant was as active as wt-SF2/ASF in promoting translation in vitro
, suggesting that RNA binding was not affected by these substitutions.
The three guanidino nitrogen atoms in the Arg side chain can potentially form five hydrogen bonds (H bonds) with H-bond acceptors in RNA, resulting in a network of H-bond interactions, which are not possible with Lys, as it has a single terminal amino group (10
). For example, a short Arg-rich basic peptide from the HIV-1 Tat protein binds specifically to the transactivation-responsive region RNA, and a Lys substitution results in a loss of binding and transactivation (15
The methylation of the two terminal amino groups in the Arg side chain does not alter the positive charge but increases the hydrophobicity, makes the side chain bulkier, and, most importantly, blocks any potential H-bond formation. This could provide a potential means of regulating protein-RNA as well as protein-protein interactions involving SF2/ASF such that the methylation of the Arg residues abolishes some interactions based on H bonding while leaving electrostatic interactions unaffected. For example, Pro-rich motifs in Sam68 interact with both SH3 and WW domains present in interacting partners; the methylation of RG repeats that flank the Pro-rich motifs reduces the binding of Sam68 to the SH3 domains of p59fyn
and phospholipase C-γ1 without affecting binding to the WW domain of FBP30 (4
Because the motif recognized by PRMT1 is not limited strictly to RGG and related sequences (60
), it is possible that other Arg residues of SF2/ASF are also modified. One precedent is the methylation of R3 in histone H4 by PRMT1, in which the methylated Arg is not part of an Arg/Gly-rich region (58
). As in the case of histones, the methylation state in the SF2/ASF linker region may control various protein-protein interactions, either directly or by influencing other modifications of SF2/ASF, such as the phosphorylation of the RS domain. Such regulation via Arg methylation, in conjunction with phosphorylation-dephosphorylation cycles of the RS domain, could play a role in the localization and trafficking of SF2/ASF between cellular compartments. A precedent for this type of cross talk was observed previously for Npl3p of budding yeast (63
). The phosphorylation of the RS domain is essential for the interaction of SF2/ASF with transportin-SR2 (TRN-SR2), which acts as a receptor for the nuclear import of SR proteins (36
). Furthermore, the phosphorylation state of SF2/ASF influences its activity as an adaptor protein for Tip-associated protein (TAP)-mediated mRNA export (27
). However, when we analyzed the interactions of wt-SF2/ASF and its mutant versions (A1
) with either TAP or TRN-SR2, we observed that all versions of SF2/ASF interacted similarly with these two proteins, arguing against the possibility that these three Arg residues, or their methylation states, affect these interactions (data not shown).
In addition to changing the properties of binding sites and affecting other modifications, methylated arginines are also involved directly in protein-protein interactions. The Tudor domain of the SMN protein interacts directly with symmetric dimethyl Arg residues in proteins with this modification (18
). However, proteins and their respective domains that may bind to asymmetric dimethyl Arg residues in SF2/ASF, and other proteins with the same modification, are yet to be discovered.
The triple-Ala mutant of SF2/ASF was unable to enhance NMD and failed to modulate the alternative splicing of endogenous target pre-mRNAs when modestly overexpressed. Furthermore, due to the accumulation of the protein in the cytoplasm, A1
was more efficient at enhancing the translation of a luciferase reporter than wt-SF2/ASF. When we restored the nuclear localization of the A1
protein by the C-terminal fusion of a nuclear retention sequence from the nonshuttling SR protein SC35 (17
), the resulting A1
-NRS1 protein was as effective in promoting NMD as wt-SF2/ASF, demonstrating that the effects observed with A1
were due to mislocalization. However, we note that at high levels of overexpression, A1
was as effective as SF2/ASF in promoting the selection of the proximal 5′ss of the β-globin model pre-mRNA with a duplicated 5′ss, most likely due to its ability to shuttle, resulting in enough protein in the nucleus to modulate alternative splicing. However, it is interesting that even at high levels of overexpression and with the ability to shuttle, A1
was inactive in promoting NMD. The ability of SF2/ASF to regulate splicing in a concentration-dependent manner is well documented (16
), whereas the precise mechanisms underlying the effect of SF2/ASF in NMD (65
) remain largely unknown, although it was shown recently that the transient overexpression of SF2/ASF promotes an increase in the efficiency of the pioneer round of translation (51
). Nonetheless, we have conclusively confirmed the initial observation (65
) that the nuclear localization of SF2/ASF is essential for its activity in enhancing NMD.
Although the detailed mechanisms through which SF2/ASF promotes transformation are understood only in part (30
), an improper cellular localization of SF2/ASF may be one of the ways through which it exerts its oncogenic activity, especially due to its regulatory roles in multiple posttranscriptional events in both the nucleus and the cytoplasm. For example, in sputum, an increase in the cytoplasmic levels of another splicing factor, hnRNP A2, serves as a powerful predictor of lung cancer almost a year prior to clinical detection (57
), suggesting that such a change may be a prerequisite for the transformation of lung epithelial cells.
Intriguingly, the K1
mutant was functionally indistinguishable from wt-SF2/ASF, pointing toward the importance of charge at these positions as opposed to the methylation state per se
. Studies of the yeast SR-like protein Npl3p (52
); other shuttling RNA-binding proteins in mammals, such as some hnRNPs (24
); and the transcription factors TAF15 and Ewing's sarcoma oncoprotein (EWS) (3
) have shown that the Arg methylation of RGG motifs is involved in controlling the nucleocytoplasmic distribution of these proteins. However, no obvious defect in the nuclear export or localization of Npl3p was observed upon the replacement of Arg residues in the RGG motifs with Lys (45
). A similar observation was made with the yeast protein Hrp1p, which resembles hnRNPs and also contains RGG motifs; but in this case, additional changes of the Arg residues to Glu or Gln resulted in cytoplasmic localization, most likely through impaired nuclear import (61
). In mammals, EWS interacts with components of both the transcriptional machinery, via its N-terminal transactivation domain, as well as the splicing machinery, via its C-terminal domain, and is thought to couple transcription and splicing (62
). EWS is methylated at two RGG boxes, RGG2 and RGG3, which are required for its nuclear localization (3
). However, only the replacement of Arg residues in the RGG boxes with Ala, and not Lys, altered the transcriptional activity of EWS (1
Our observation that at least one of the substituted lysines (R109K) in the K1K2K3 version of SF2/ASF was dimethylated suggests that both the methylation state and the positive charge at these positions may contribute to the localization of SF2/ASF. Although it is possible that naturally occurring Lys residues in SF2/ASF are methylated, we had not expected to find methylation of the substituted Lys residues. Also, in the absence of quantitative mass spectrometry data, we do not know what fraction of the total K1K2K3 protein expressed in 293E cells underwent this modification, so it is possible that dimethylation of the substituted Lys109 is present in only a small fraction of the protein.
Experiments involving the SM and the KM mutants (see Fig. S2 in the supplemental material), which had activities similar to those of wt-SF2/ASF, did not conclusively solve the conundrum of charge versus methylation due to the possibility that even in the absence of the glycines in the RGG/GRG motifs, the Arg residues in the SM mutant might still be methylated by PRMT1 and/or other methyltransferases. Furthermore, the treatment of HeLa cells with the specific PRMT1 inhibitor AMI-1 did not alter the localization of either endogenous SF2/ASF or transfected wt-SF2/ASF and K1K2K3 proteins, consistent with the importance of positive charge (data not shown). However, PRMT1 methylates many proteins in the cell; therefore, blocking its activity may affect various cellular processes, including nucleocytoplasmic trafficking, which could account for the observed negative result. Another possibility is that methylation of the three arginines in SF2/ASF may also involve other methyltransferases, which may be active when PRMT1 is inhibited.
In summary, our findings underscore the importance of the proper localization of SF2/ASF for its activity in key nuclear and cytoplasmic processes. We have further identified the signals that control the distribution of SF2/ASF between nucleus and the cytoplasm and also generated mutants of SF2/ASF with a partial loss of function. Such mutants will prove useful in future studies to dissect the mechanisms through which SF2/ASF affects various normal cellular processes as well as oncogenic transformation.