Beside its long established role in translation

Beside its long-established role in translation, eIF4E was recently shown to participate in further post-transcriptional functions as nuclear export and mRNA decay pathways [19]. Culjkovic et al. presented a list of mRNAs that are targets for eIF4E in the nucleus and may be transported in an eIF4E-dependent manner. These findings include for example the cyclin D1 mRNA, which seems to be exported via a not yet clarified mechanism involving eIF4E and a newly identified structural element in the cyclin D1 mRNA 3′-UTR [20]. However, the adapter proteins for the CRM1-mediated nucleocytoplasmic transport of many other mRNAs are still unknown. The inducible isoform of the nitric oxide synthase (iNOS) is an enzyme involved in many physiologic and pathophysiologic pathways like wound healing, inflammation, and cancer [21], [22]. The regulation of human iNOS expression depends on transcriptional and post-transcriptional mechanisms [22], [23]. The iNOS mRNA 3′-UTR contains five AREs displaying destabilizing properties [24]. In the last years we identified a complex network of RNA binding proteins that interact with these AREs and modulate iNOS mRNA stability. The most important destabilizing factors in this network are the KH-type splicing regulatory protein (KSRP) [25] and the four isoforms of the AU-binding factor 1 (AUF-1, also known as hnRNP D) whereas HuR and the polypyrimidine-tract binding protein (PTB) stabilize the human iNOS mRNA [24], [26]. In Simeprevir synthesis to its common destabilizing function, TTP also leads to stabilization of the iNOS mRNA without binding to it [27].
Material and methods
Results
Discussion This study demonstrates that, beside modulation of mRNA stability, control of nucleocytoplasmic mRNA transport is another important facet in the post-transcriptional mechanisms involved in human iNOS expression. While the bulk of poly(A)+ mRNAs is exported from the nucleus via the complex of TAP/Nxt-proteins, there are some exceptions that are transported from the nucleus to the cytoplasm in a CRM1-dependent manner. Most but not all mRNAs exported in a CRM1-dependent fashion contain AU-rich elements (AREs) in their 3′-UTRs [10]. To assess which export pathway is used by the iNOS mRNA, we inhibited CRM1 activity by incubation of DLD-1 cells with LMB. Since LMB covalently and exclusively binds to CRM1, it represents a potent and specific inhibitor of the CRM1 transport activity [38]. Inhibition of CRM1 activity as well as siRNA-mediated reduction of CRM1 expression leads to a marked decrease in iNOS expression and activity (Fig. 1, Fig. 2). LMB mediated inhibition of iNOS expression has also been described in murine BV2 microglial cells [11]. Therefore, CRM1 seems also to be involved in the regulation of human iNOS expression. Since we observed no influence of LMB on human 16kb iNOS promoter activity (Fig. 1D) and CRM1 is known to mediate the transport of RNAs from the nucleus to the cytoplasm, we also analyzed the amount of cytoplasmic and nuclear iNOS mRNA after LMB treatment of the cells (Fig. 3). The ratio of cytoplasmic to nuclear iNOS mRNA is clearly decreased after CRM1 inhibition, indicating that the export of the iNOS mRNA is, at least in part, accomplished by a CRM1-dependent mechanism. We observed similar data for the nucleocytoplasmic transport of the TNF-α and TTP mRNA (data not shown). In contrast to the cytokine-regulated ARE-containing iNOS mRNA, the amount of cytoplasmic and nuclear GAPDH mRNA remains unchanged after CRM1 inhibition. This indicates that CRM1-mediated mRNA export actually is only perceived by a specific class of mRNAs, most likely those that contain AREs in their 3′-UTR and whose expression is regulated cytokine-dependently. This assumption parallels the observation of Schutz et al. that CRM1-mediated RNA transport only occurs in activated T cells [13].