| Nuclear RNA export is a receptor-mediated pathway in eukaryotic cells. Karyopherin proteins are a family of nucleocytoplasmic transport receptors, mediating the nuclear export of proteins, non-coding RNA species and a few mRNA molecules. However, bulk mRNA is exported by Tap, which is not a member of the karyopherin family. We established a heterologous RNA tethering system to quantitatively measure the ability to export tethered mRNA molecules by transport receptors. We demonstrated that the karyopherin proteins, as well as the Tap protein, are capable of exporting tethered mRNA molecules to the cytoplasm.; HIV-1 mRNA transcripts are exported by the viral Rev protein via Crm1, a member of the karyopherin family. We showed that the direct recruitment of Crm1 to viral mRNA molecules, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins. Thus, we determined that the sole role of the viral Rev protein is to recruit Crm1 to the viral mRNA transcripts.; Finally, we identified Exportin-5, a member of the karyopherin family, as the nuclear export factor for pre-microRNAs. Exportin-5 binds pre-microRNAs specifically and directly in vitro, in a RanGTP dependent manner. Knocking down Exportin-5 inhibited pre-microRNA nuclear export, and therefore, the expression of mature microRNAs in the cytoplasm. Furthermore, we compared the effect of overexpressing key factors in the microRNA biogenesis pathway and concluded that Exportin-5 is the rate-limiting factor in the pathway.; Together, these results improved our understanding of karyopherin-mediated nuclear RNA export pathways and proved that Exportin-5 is the essential nuclear export factor for pre-microRNAs, thereby providing new insights into the microRNA biogenesis pathway. |
