Serine͞arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine͞serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin ␤ family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains. S erine͞arginine-rich proteins (SR proteins) comprise a large family of eukaryotic pre-mRNA splicing factors that contain a discrete domain rich in arginine͞serine (RS) dipeptide repeats (RS domain). SR proteins are essential for pre-mRNA splicing and also play a pivotal role in determining alternative splice site selection (1-3). Cytological studies reveal that SR proteins are primarily localized in nuclear speckles-structures that serve as the sites for storage and͞or reassembly of splicing factors. However, some, but not all, SR proteins continuously shuttle between the nucleus and the cytoplasm (4). Subcellular localization of SR proteins is influenced by their phosphorylation state and by the level of transcription activity within the cells (4-8). Hyperphosphorylation of the RS domain causes relocalization of SR proteins from nuclear speckles to a more diffuse distribution in the nucleoplasm or causes their cytoplasmic accumulation (4-8). When mRNA synthesis is blocked, the speckled pattern of some SR proteins becomes more prominent and, nevertheless, a subset of shuttling SR proteins is retained in the cytoplasm (4). Therefore, both the nucleocytoplasmic transport and subnuclear localization of SR proteins are likely to be complex and regulated processes, the underlying mechanisms for which remain to be delineated.Nucleocytoplasmic transport of macromolecules via the nuclear pore complex (NPC) is generally mediated by saturable transport receptors that recognize specific signals within proteins (9-12). Nuclear import of proteins with a classical basic nuclear localization signal (NLS) is mediated by the dimeric complex of importin...

Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-␤ family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins. SR1 proteins are a superfamily of eukaryotic proteins that contain repetitive serine-arginine dipeptides in a domain known as RS domains (1-3). SR proteins are primarily involved in the splicing of precursor mRNA. Some SR splicing factors are essential for pre-mRNA splicing, and some can act as crucial players in alternative splicing by modulating splice site choice. A group of SR proteins that can be precipitated by magnesium and recognized by monoclonal antibody (mAb) 104 play both essential and regulatory roles in pre-mRNA splicing (4). Each of these SR proteins can complement splicing-deficient cytoplasmic S100 extracts as well as affect splice site selection at elevated concentrations (1-3). In addition to splicing factors, RS domains are present in other proteins, such as a group of human papillomavirus E2 transcriptional activators (5, 6), RNA pol II-associated SR-like proteins (7), transcriptional coactivator PGC-1 (8), and pre-mRNA cleavage factor Im (9). Thus, RS domain-containing proteins can function in gene expression at different levels.Cytological studies have revealed that a variety of SR proteins are localized in nuclear speckled domains, which are thought to be the sites for storage/reassembly of splicing factors and/or supplying splicing factors to active genes (10, 11). The RS domains of some, but not all, SR proteins have been shown to be necessary and sufficient for targeting to the nuclear speckles (12, 13). Hyperphosphorylation of the RS domain by SR protein-specific kinases can relocate SR proteins from a speckled pattern into a more diffuse distribution (14 -16). Recent evidence indicates that a subset of SR proteins can continuously shuttle between the nucleus and the cytoplasm (17). The phosphorylation states of the RS domain appear to have...

The multicomponent exon junction complex (EJC) is deposited on the spliced mRNA during pre-mRNA splicing and is implicated in several post-splicing events, including mRNA export, nonsensemediated mRNA decay (NMD), and translation control. This report is the first to identify potential post-translational modifications of the EJC core component Y14. We demonstrate that Y14 is phosphorylated at its repeated arginine/serine (RS) dipeptides, likely by SR protein-specific kinases. Phosphorylation of Y14 abolished its interaction with EJC components as well as factors that function downstream of the EJC. A non-phosphorylatable Y14 mutant was equivalent to the wild-type protein with respect to its association with spliced mRNA and its ability in NMD activation, but the mutant sequestered EJC and NMD factors on ribosome-containing mRNA ribonucleoproteins (mRNPs). We therefore hypothesize that phosphorylation of Y14 occurs upon completion of mRNA surveillance, leading to dissociation of Y14 from ribosome-containing mRNPs. Moreover, we found that Y14 is possibly methylated at multiple arginine residues in the carboxyl-terminal domain and that methylation of Y14 was antagonized by phosphorylation of RS dipeptides. This study reveals antagonistic post-translational modifications of Y14 that may be involved in the remodeling of Y14-containing mRNPs.Eukaryotic mRNAs undergo several processing steps before export to the cytoplasm for translation. The splicing reaction removes introns from precursor mRNAs (pre-mRNAs) 4 and positions the exon junction complex (EJC) on spliced mRNA in a sequence-independent manner (1, 2). The EJC is a dynamic multicomponent complex consisting of a heterodimer of Y14 with Mago and a number of associated factors (1, 2). The EJC may be functionally connected to transcription and acts as an adaptor for recruiting factors involved in the RNA metabolism steps downstream of splicing (3).Previous work suggests that the EJC functions for the nuclear export of spliced mRNAs via the interaction of Y14/Mago, as well as other components, with the mRNA export receptor TAP (4 -6). However, depletion of EJC components only marginally affects bulk poly(A) ϩ RNA export in cultured Drosophila cells (7), suggesting that the EJC may be an accessory factor for mRNA export. On the other hand, Y14/ Mago and RNPS1, another EJC component, directly promote NMDmediated mRNA degradation (8, 9). Y14 interacts with the NMD initiator Upf3 in the nucleus, which subsequently recruits other Upf proteins to yield the active NMD complex (1, 2). Depletion of Y14 abolishes NMD, indicating its essential role in this pathway (10). Recent reports show that the EJC promotes efficient translation by enhancing polysome association with mRNAs (11, 12). In particular, Y14/Mago, as well as RNPS1, is implicated in this translation enhancement (11-13). Thus, the EJC participates in several post-splicing events, including mRNA export/surveillance and translation control (1, 2, 14). The Y14/ Mago heterodimer acts as a core that interacts with sever...

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5 splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A) ؉ RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.In eukaryotic cells, the step to remove the intron sequences from the pre-mRNA is carried out by the spliceosome, a macromolecular complex consisting of small nuclear ribonucleoproteins (snRNPs) and a number of protein factors (22). A family of Ser/Arg-dipeptide-rich proteins (SR proteins) play essential roles in constitutive splicing and/or can modulate alternative splice site selection (13). Atypical SR protein RNPS1 was previously characterized with a splicing activity that promotes utilization of distal alternative 3Ј splice sites (32). However, recombinant RNPS1 instead synergizes with prototypical SR proteins to activate both constitutive and alternative pre-mRNA splicing, suggesting the role of RNPS1 as a general splicing activator (32). On the other hand, RNPS1 associates with SAP18 and acinus proteins to form the apoptosis and splicing-associated protein (ASAP) complex, which inhibits in vitro splicing and promotes apoptosis (42). It appears that RNPS1 functions to activate or suppress splicing by forming complexes with different regulatory proteins.During the pre-mRNA splicing process, a multiprotein complex is deposited on the spliced mRNP (24,25). This complex occupies a region 20 to 24 nucleotides (nt) upstream of the splice junctions of mature mRNA and is thus termed the exonexon junction complex (EJC) (25). RNPS1 has been determined as a component of the EJC (25), which is consistent with the observation that RNPS1 specifically associates with spliced mRNAs in vitro (32). The EJC is thought to function as an adaptor platform that provides multiple postsplicing functions (26). This notion is apparently held true in the case of nonsense-mediated decay (NMD), which subjects aberrant mRNAs with premature termination co...

Members of the serine/arginine-rich (SR) protein family play an important role in both constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich domain (RS domain) can modulate the activity and the subcellular localization of SR proteins. However, whether the SR protein family members are individually regulated and how this is achieved remain unclear. In this report we show that 5,6-dichloro-1 beta-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II-dependent transcription, specifically induced hyperphosphorylation of SRp55 but not that of any other SR proteins tested. Hyperphosphorylation of SRp55 occurs at the RS domain and appears to require the RNA-binding activity. Upon DRB treatment, hyperphosphorylated SRp55 relocates to enlarged nuclear speckles. Intriguingly, SRp55 is specifically targeted for degradation by the proteasome upon overexpression of the SR protein kinase Clk/Sty. Although a destabilization signal is mapped within the C-terminal 43-amino acid segment of SRp55, its adjacent lysine/serine-rich RS domain is nevertheless critical for the Clk/Sty-mediated degradation. We report for the first time that SRp55 can be hyperphosphorylated under different circumstances whereby its fate is differentially influenced.