RNA location and modeling of a WD40 repeat domain within the vault

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, compos ed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vau lt has been shown to affect multidrug resistance in cancer cells, and one p articular component, MVP, is thought to play a role in the transport of dru g from the nucleus. To locate the position of the vRNA, Vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the r esulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a PEA-resolution structure. Diffe rence mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vau lt caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric c onsiderations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mism atch between the barrel and the caps. The molecular architecture of the com plex is emerging, with 96 copies of MVP composing the eightfold symmetric b arrel, and the VRNA together with one copy of TEP1 and four predicted copie s of VPARP comprising each cap.