We and others have previously shown that SM plays multiple roles in posttranscriptional processing of EBV mRNA and cellular mRNA (2
). Although the target sequences for SM binding have not been biochemically defined, abundant evidence indicates that SM contacts RNA directly. SM can be covalently cross-linked to mRNAs in vitro
and exhibits RNA-binding specificity when immunoprecipitated from EBV-infected cells undergoing lytic replication (14
). Further, SM enhances the expression of EBV mRNAs preferentially, with some genes being SM dependent for expression, while others are SM independent (13
). The posttranscriptional effect of SM in enhancing mRNA accumulation has been attributed to an ability to act as a nuclear export factor. According to this model, SM binds to RNA via an RBD and recruits Ref/Aly, TAP, or CRM1 cellular export proteins (2
). SM also enhances the nuclear accumulation of target mRNAs posttranscriptionally, suggesting that SM stabilizes mRNA, similar to its KSHV homolog ORF57 (25
SM also has distinct effects on spliced mRNA transcripts. SM inhibits the expression of spliced cellular targets in transfection assays (31
). Recently, we demonstrated that SM inhibits expression of the spliced immediate-early EBV transactivator Rta (39
). Inhibitory effects of SM on spliced gene expression have been attributed to premature export of unspliced pre-mRNAs by SM, analogous to the mechanism of HIV Rev protein (4
). However, the inhibitory effect of SM on spliced gene expression is target specific, suggesting that SM interacts specifically with the process of splicing (25
). Our recent demonstration that SM can affect splice-site selection lends further support to the hypothesis that SM exerts direct effects on splicing in the nucleus (40
). The interaction of SM with SRp20 and the involvement of this association in alternative splice site selection suggest a model whereby SM binds to specific pre-mRNA sites and recruits SRp20 via protein-protein interactions (Fig. ). The RS domain of SRp20 would then direct the formation of spliceosome assembly by recruiting snRNPs and potentially by contacting RNA at discrete sites in the intron (33
). In this manner, SM could utilize SRp20 and co-opt its functions of directing splice-site selection even at sites of mRNA devoid of SRp20 binding sites. Recruitment of SRp20 by SM would thereby enable usage of a set of novel splice sites to generate mRNA isoforms unique to cells in which EBV is replicating lytically.
FIG. 7. SM recruitment of SRp20 to pre-mRNA. SM is shown binding to pre-mRNA at an SM binding site near a 5′ exon to facilitate splicing at a potential alternative splice site. The SM response element (RNA binding site) is shown as a cross-hatched box. (more ...)
SRp20 is important for export of intronless histone mRNAs and is thought to act as an export factor by virtue of its ability to bind RNA via its RBD and undergo nucleocytoplasmic shuttling (19
). SRp20 also interacts with the export mediator TAP via a region in the central portion of the SRp20 molecule (15
). Interestingly, this region overlaps with the SM-binding region of SRp20. Thus, it is possible that binding to SM may interfere with SRp20's ability to enhance export of its own RNA cargoes. Alternatively, SRp20 binding to SM may be compatible with TAP recruitment in a manner analogous to the process whereby Ref/Aly, a cellular RNA-binding protein, is thought to bind mRNA and then hand off the mRNA to TAP (16
). The net effect of the interaction of SM with SRp20 on export of cellular histone mRNAs and other RNAs remains to be determined. Herpes simplex virus (HSV) ICP27, a homolog of EBV SM, has been shown to inhibit splicing by altering the function of SRPK1, an SR protein kinase, leading to hypophosphorylation of SRp20 and other SR proteins (34
). Recently, SRp20 has been implicated in the export of HSV intronless mRNAs (8
). Because hypophosphorylation of SRp20 is linked to its ability to export RNA (19
), ICP27 may facilitate HSV RNA export by SRp20. Whether SRp20 plays a role in enhancing intronless EBV mRNA stabilization and export is another important question that is raised by these findings. The net effect of the SRp20-SM interaction on EBV mRNA expression is likely to be complex, since the SRp20 effects may also be RNA target dependent.
In summary, we have shown that SM protein binds SRp20 independently of RNA, via discrete protein-protein interactions, and that the interaction is important for the ability of SM to direct specific alternative splice-site selection. Further, recruitment of SRp20 by SM suggests a model by which SM may utilize the RNA and spliceosome-recruiting properties of SR proteins to redirect these proteins to convert SM-binding sites to splicing enhancers. These findings implicate mRNA splicing as another target pathway by which EBV modulates the cellular milieu during lytic replication. Inasmuch as >70% of cellular genes undergo alternative splicing (21
), it is likely that EBV affects a number of genes in addition to those currently described, thereby altering the “splicing code” of the cell to facilitate EBV replication.