| Nuclear export of SRP: Once SRP assembles in the nucleus, the RNA-protein complex must be exported to the cytoplasm. Work in other labs suggests that the multipurpose nuclear export factor Xpo1p/CRM1 is responsible for SRP export. However, it is not known how CRM1 interacts with SRP. Using a Xenopus oocyte microinjection assay, we found that CRM1 likely interacts with SRP54 to mediate export, since export on SRP RNA injected into the nucleus was stimulated by co-injection of SRP54 and inhibited by leptomycin, a drug that prevents CRM1 binding to its substrates (Figure 1). | |
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Figure 1. CRM1-dependent, SRP19-SRP54 stimulation of SRP RNA nuclear export. A mixture of labeled RNAs was injected into the nucleus (N) without (no add) or with various proteins. C=RNA in cytoplasm after two hours, T=total injected. Lanes 5, 6 reveal little to no export; export was stimulated by co-injection of SRP19+ SRP54 (lanes 8, 9); SRP19 is co-injected because SRP54 can bind SRP RNA only if SRP19 is first bound to the RNA. Stimulation was reduced by the CRM1 inhibitor LMB (lanes 14, 15). |
| We have also investigated the binding of SRP54 to CRM1 in vitro. Using truncated versions of SRP54, we mapped the CRM1 binding site to SRP54 residues 296-323, which contains a perfect match to a CRM1 consensus recognition sequence. This CRM1 binding site is located between the two major functional domains of SRP54 (G and M); in the bacterial counterpart of SRP54, this segement forms a flexible, exposed loop, suggesting that the CRM1 binding site in mammalian SRP should be exposed and accessible. Also, we measured the affinity of CRM1 for SRP54 and find that the proteins interact with nanomolar affinity (Figure 2). | |
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Figure 2. SRP54 is a CRM1 ligand. A. Full-length and portions of SRP54 (left) were immobilized on Sepharose and incubated with CRM1. Bound proteins were eluted and subjected to gel electrophoresis (right). The smallest segment of SRP54 to bind CRM1 was residues 296-323. B. In the presence of a CRM1 ligand, CRM1-bound RanGTP is resistant to RanGAP; a concentration of CRM1 giving half-maximal GTP hydrolysis in the presence of saturating ligand corresponds to the Kd for ligand binding. Current questions: The evidence that CRM1-SRP54 mediates SRP nuclear export in mammalian cells is indirect. Experiments using RNA interference and immunolocalization will be carried out to obtain direct evidence for the role of these proteins in SRP nuclear export. |