Using a Xenopus oocyte microinjection assay, we have shown that nuclear export of SRP RNA is a facilitated process, implying the existence of one or more nuclear transport factors that recognize SRP RNA or an SRP RNA-protein complex. Furthermore, we found that a mutant SRP RNA unable to bind SRP9 and SRP14 was transported at a reduced rate, implying that these two proteins have a nuclear role in RNA export. Thus, we proposed that some or all of the SRP proteins enter the nucleus to assemble with SRP RNA and it is this complex that is the substrate for export. Xenopus oocytes are ideal for investigating RNA nuclear export. Following microinjection of a substrate or transport inhibitor, the nucleus can be quickly separated manually from the remainder of the cell to quantitate the amount of material in each compartment.
Arrows show isolated nuclei.
Figure courtesy of Dr. Brian Kay, Univ. In vitro nuclear import assays are being used to assay the ability of each of the six SRP proteins to enter the nucleus in a facilitated manner. The same assays, in conduction with purified factors, are being used to identify the import receptors for SRP proteins. Total cell extracts are being incubated with immobilized purified SRP proteins to identify potential transport factors. Proteins which bind the SRP proteins will be identified with antibodies to known transport factors and also assayed in vitro assays for their ability to promote transport.