Date Published: November 18, 2009
Publisher: Public Library of Science
Author(s): Isabelle Ote, Marielle Lebrun, Patricia Vandevenne, Sébastien Bontems, Cahora Medina-Palazon, Evelyne Manet, Jacques Piette, Catherine Sadzot-Delvaux, Juan Valcarcel. http://doi.org/10.1371/journal.pone.0007882
Abstract: Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.
Partial Text: In eukaryotic cells, export of mRNAs from the nucleus into the cytoplasm is a complex and well regulated process. In metazoans, mature mRNPs are transported by the essential mRNA export receptor TAP/NXF1 that shuttles between the nucleus and the cytoplasm and escorts competent mRNPs out of the nucleus through direct interactions with nucleoporins lining the nuclear pore . Because of its low affinity for binding mRNAs, TAP/NXF1 needs export adaptor proteins to interface with mature transcripts that are ready for export. So far, the best-characterized adaptor of TAP/NXF1 is the Aly/REF protein . For its recruitment to mRNAs, Aly/REF requires the essential mRNA export factor UAP56  and these two proteins were originally found to be associated with the exon junction complex (EJC) formed during late stage of pre-mRNA splicing . More recent studies have shown that UAP56 and Aly/REF are part of the multi-protein TREX (transcription-export) complex, which is recruited co-transcriptionally to the 5′ end of mRNAs via the cap-binding protein Cbp80  and is essential for the export of both spliced and intronless mRNAs , . While UAP56 was shown to be essential for mRNA export in both Drosophila and C. elegans, Aly/REF seems to be dispensable, suggesting the existence of additional mRNA export adaptors , . Moreover, it has been speculated that the shuttling SR proteins SRp20, 9G8 and ASF/SF2, retained on mRNAs, might also generate export-competent mRNPs. Interestingly, shuttling SR proteins have been shown to promote export of both intronless  and intron-containing  mRNAs. Thus, these proteins may be export adaptors shared by different mRNA classes. This hypothesis is supported by the fact that, like the adaptor Aly/REF, shuttling SR proteins can directly interact with TAP/NXF1 and can recruit this export receptor to bound mRNAs .
Available data suggest that IE4 acts as an important regulator on VZV and cellular genes expression and could exert its functions at a post-transcriptional level. However, the molecular mechanisms are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. The main results obtained in this work can be summarized as follows: (i) In yeast two-hybrid, the IE4 RbRc domain interacts with cellular proteins involved in mRNA metabolism. In a cellular context, IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20, and this interaction is not bridged by RNA. (ii) IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in vitro on residue Ser-136. (iii) IE4 is able to bind RNA in vitro and the presence of RNA stabilizes complexes containing IE4, TAP/NXF1 and Aly/REF. (iv) IE4 influences export of mRNAs through the TAP/NXF1 pathway.