Our current results suggest that nuclear import of Npl3p is affected by juxtaposed methylation and phosphorylation ( a). Our finding that Sky1p-mediated phosphorylation plays an important role in nuclear import is consistent with the localization of the kinase in the cytoplasm. Thus, newly synthesized Npl3p may be readily phosphorylated by Sky1p in the cytoplasm for efficient interaction with its nuclear import receptor Mtr10p, which leads to efficient nuclear import, and this modification may also be important for the function of Npl3p in the nucleus. Upon translocation to the nucleus, RanGTP and RNA release Npl3p from the import complex, as previously demonstrated (Senger et al. 1998
). The arginine methyltransferase Hmt1p/Rmt1p is localized in the nucleus even when it is overexpressed (Henry and Silver 1996
), which may mediate multiple steps important for Npl3p export with RNA as proposed based on genetic studies (Shen et al. 1998
). Because Npl3p is localized in the nucleus at steady state, its import ( a, large arrow) must be faster than export ( a, medium arrow) in wild-type yeast.
Figure 9 A model for Npl3p nuclear import and export controlled by Sky1p-mediated phosphorylation and Hmt1p-mediated methylation. (a) In wild-type cells, Sky1p in the cytoplasm phosphorylates Npl3p, which is required for its efficient nuclear import by Mtr10p. (more ...)
Our results also extend previous genetic data regarding the function of methyltransferase Hmt1p/Rmt1p in Npl3p nucleocytoplasmic shuttling. As reported previously (Lee et al. 1996
), wild-type GFP-Npl3p accumulated in the cytoplasm of nucleoporin mutant (nup49–313
) cells when nuclear import was impaired ( b, small arrow) while nuclear export proceeded normally ( b, medium arrow). In the present studies, we found that phosphorylation defects also caused the accumulation of mutant npl3p in the cytoplasm of wild-type yeast (see ; illustrated in c) due to its inefficient interaction with Mtr10p. The function of Hmt1p/Rmt1p became evident when the HMT1/RMT1
gene was deleted or inactivated in these genetic backgrounds ( and ). When HMT1/RMT1
was deleted in the nup49–313
strain, wild-type GFP-Npl3p quantitatively relocated to the nucleus, as reported previously (Shen, et al. 1998). The observation was previously interpreted to indicate that nuclear export of GFP-Npl3p was impaired in the absence of Hmt1p/Rmt1p ( d, small arrow). However, considering the possibility that nuclear import in nup49–313
cells was impaired but not completely blocked at the restrictive temperature, redistribution of Npl3p to the nucleus of hmt1/rmt1
Δ cells could also be contributed by the removal of the methylation interference of phosphorylation as shown in this study. As a result, improved import ( d, medium arrow) was concomitant with impaired export in the absence of Hmt1p/Rmt1p, which together caused efficient shift of Npl3p back into the nucleus.
According to this model, one would predict that the improvement of nuclear import could not occur if the Sky1p phosphorylation site is mutated. Indeed, we observed that GFP-npl3p(S411A) expressed from a plasmid under the GAL1
promoter remained in the cytoplasm of hmt1/rmt1
Δ cells (data not shown). Consistently, endogenously expressed npl3p(E409K) only partially shifted back to the nucleus when Hmt1p/Rmt1p was inactivated (McBride et al. 2000
). Thus, the observed distribution of the mutant npl3p in both the cytoplasm and the nucleus is likely due to defects in both import and export pathways (indicated by small arrows in both directions in e). Together, these observations in combination with biochemical evidence presented in this report lend strong support for a positive role for the major arginine methyltransferase in Npl3p export and a negative role for this enzyme in Npl3p import in budding yeast.
The model presented in raises several fundamental questions with regard to the functional consequences of phosphorylation and arginine methylation: (a) Which components of this regulatory pathway are conserved in mammalian cells? (b) Are phosphatases and demethylases involved in the pathway? and (c) Does this regulatory pathway represent some of the key steps in controlling the function of shuttling RNA binding proteins? Below, we discuss the significance of our novel findings in the context of these global questions.
The NLS in Npl3p is composed of both repetitive (RGG repeats) and nonrepetitive (Sky1p phosphorylation site) sequences. Interestingly, these sequence features are conserved in two separate classes of RNA binding proteins (hnRNP and SR proteins) in mammalian cells (Burd and Dreyfuss 1994
; Fu 1995
). Many hnRNP proteins shuttle between the nucleus and the cytoplasm, but their transport appears to be mediated by separate signal sequences adjacent to their RGG domains (Nakielny and Dreyfuss 1997
). Although hnRNP proteins are extensively modified by arginine methylation in mammalian cells (Liu and Dreyfuss 1995
), how methylation might modulate their transport signals remains to be addressed. The RS domain in SR proteins is critical for nuclear and subnuclear localization (Li and Bingham 1991
; Hedley et al. 1995
; Cáceres, et al. 1997). It was recently shown that SR proteins in mammalian cells interact with two Mtr10p-related receptors called transportin-SR (Kataoka et al. 1999
) and transportin-SR2 (Lai et al. 2000
). Thus, the transport machinery for SR and SR-like proteins, including nuclear import receptors (transportin-SR, transportin-SR2 and Mtr10p) and regulators (SRPKs), are conserved between yeast and humans. However, the yeast and metazoan receptors have evolved distinct substrate specificities because Mtr10p contacts the RGG box in Npl3p whereas transportin-SR interacts with the RS domain containing SR/RS instead of RGG repeats. Furthermore, it appears that transportin-SR can interact with unphosphorylated SR proteins in vitro, although potential phosphorylation regulation of the interaction was not addressed (Kataoka et al. 1999
). On the other hand, the interaction between SR proteins and transportin-SR2 was shown to be dependent on SRPK-mediated phosphorylation (Lai et al. 2000
). Future studies will define the signal sequences for nuclear import of SR proteins and address how their nuclear import might be regulated by SRPKs in mammalian cells.
Phosphorylation of SR proteins is required for spliceosome assembly and dephosphorylation is critical for the splicing reaction to occur (Mermoud et al. 1994
). Npl3p and other Sky1p substrates may play a role in pre-mRNA splicing in budding yeast, but it is not known whether a phosphorylation–dephosphorylation cycle accompanies their function in the nucleus. In contrast to reversible phosphorylation, the reversibility of arginine methylation remains highly controversial. It is clear that arginine methylation is rather stable (Desjardins and Morell 1983
). It has been argued that demethylation may be absent because the reaction to break the N–C bond would be energetically unfavorable and a potential arginine demethylase has not been found. In the absence of a specific arginine demethylase, we speculate that the stable arginine modification may serve as a mechanism to allow shuttling Npl3p sufficient time in the cytoplasm to unload its RNA cargo. Whether Sky1p-mediated phosphorylation, although inefficient on methylated Npl3p, serves as a molecular switch for unloading and reimport is another interesting possibility to be investigated. Considering the irreversibility of this modification, our data clearly demonstrated that methylation is not saturated on Npl3p, indicating that arginine methylation may be more dynamic than previously assumed. Since Npl3p is largely cytoplasmic when hypermethylated, it is possible that nuclear import of Npl3p may gradually decrease as methylation gradually accumulates. Thus, methylation may function as a molecular device to determine how many cycles each Npl3p molecule can shuttle between the nucleus and cytoplasm before it is degraded in the cytoplasm. Further experiments will test this interesting possibility.
Continuous shuttling of RNA binding proteins may not only reflect their function in transporting materials out of the nucleus, but also represent a mechanism to regulate their function in the nucleus. For example, hnRNP A1 and SR proteins can switch splice site selection in opposite ways in a concentration-dependent manner (for reviews see Fu 1995
; Manley and Tacke 1996
; Cáceres and Krainer 1997
). Therefore, the ratio of hnRNP and SR proteins in the nucleus may be critical for specific alternative splicing events. Modification of hnRNP proteins by arginine methylation and SR proteins by phosphorylation may effectively control their trafficking and therefore their ratio in the nucleus during development or in response to external stimuli. Our results using yeast as a genetic system have revealed a novel function of the conserved SRPK in regulated nuclear import and established a framework to investigate the function of the SRPK family members in mammalian cells.