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The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N -(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N -acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL)., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Vault particles are the largest naturally occurring ribonucleoprotein complexes found in the cytoplasm. In all 78 copies of major vault protein (MVP) assemble on polyribosome templates, forming recombinant vault particles, which are of great interest as encapsulation carriers for therapeutics delivery and enzyme stabilization. Baculovirus-insect cell expression is the only system that has been developed for recombinant vault synthesis, but it has low scalability and slow production rate. In this study, we demonstrated the first use of yeast cells for the production of vault particles with full integrity and functionality solely by expressing the complementary DNA (cDNA) encoding MVP. Vaults synthesized in Pichia pastoris yeast cells are morphologically indistinguishable from recombinant vault particles produced in insect cells, and are able to package and stabilize enzymes resulting in improved longevity and catalytic efficiency. Thus, our results imply that the yeast system is an economical alternative to insect cells for the production of recombinant vaults. The consistency of vault morphology between yeast and insect cell systems also underlines that polyribosome templating may be conserved among eukaryotes, which promises the synthesis and assembly of recombinant human vault particles in other eukaryotic organisms., (© 2018 Wiley Periodicals, Inc.)

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Natural vault nanoparticles are ribonucleoprotein particles with a mass of 13 MDa that have been found in a wide variety of eukaryotes. Empty recombinant vaults are assembled from heterologously expressed Major Vault Protein (MVP), forming the barrel-shaped vault shell. These structures are morphologically indistinguishable from natural vault particles. Here, we describe the packaging and purification of exogenous proteins into these recombinant vault particles by mixing with proteins attached to the INT domain that binds to MVP.

Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (K Dapp  = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.

Vault nanoparticles represent promising vehicles for drug and probe delivery. Innately found within human cells, vaults are stable, biocompatible nanocapsules possessing an internal volume that can encapsulate hundreds to thousands of molecules. They can also be targeted. Unlike most nanoparticles, vaults are nonimmunogenic and monodispersed and can be rapidly produced in insect cells. Efforts to create vaults with modified properties have been, to date, almost entirely limited to recombinant bioengineering approaches. Here we report a systematic chemical study of covalent vault modifications, directed at tuning vault properties for research and clinical applications, such as imaging, targeted delivery, and enhanced cellular uptake. As supra-macromolecular structures, vaults contain thousands of derivatizable amino acid side chains. This study is focused on establishing the comparative selectivity and efficiency of chemically modifying vault lysine and cysteine residues, using Michael additions, nucleophilic substitutions, and disulfide exchange reactions. We also report a strategy that converts the more abundant vault lysine residues to readily functionalizable thiol terminated side chains through treatment with 2-iminothiolane (Traut's reagent). These studies provide a method to doubly modify vaults with cell penetrating peptides and imaging agents, allowing for in vitro studies on their enhanced uptake into cells.

Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymatic biodegradation is an attractive technology for in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth and often have higher biodegradation rates. However, the limited stability of extracellular enzymes remains a major challenge for practical applications. Encapsulation is a recognized method to enhance enzymatic stability, but it can increase substrate diffusion resistance, lower catalytic rates, and increase the apparent half-saturation constants. Here, we report an effective approach for boosting enzymatic stability by single-step packaging into vault nanoparticles. With hollow core structures, assembled vault nanoparticles can simultaneously contain multiple enzymes. Manganese peroxidase (MnP), which is widely used in biodegradation of organic contaminants, was chosen as a model enzyme in the present study. MnP was incorporated into vaults via fusion to a packaging domain called INT, which strongly interacts with vaults' interior surface. MnP fused to INT and vaults packaged with the MnP-INT fusion protein maintained peroxidase activity. Furthermore, MnP-INT packaged in vaults displayed stability significantly higher than that of free MnP-INT, with slightly increased Km value. Additionally, vault-packaged MnP-INT exhibited 3 times higher phenol biodegradation in 24 h than did unpackaged MnP-INT. These results indicate that the packaging of MnP enzymes in vault nanoparticles extends their stability without compromising catalytic activity. This research will serve as the foundation for the development of efficient and sustainable vault-based bioremediation approaches for removing multiple contaminants from drinking water and groundwater.

We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems.

Vaults are naturally occurring nanoparticles found widely in eukaryotes. The particles can be produced in large quantities and are assembled in situ from multiple copies of the single structural protein following expression. Using molecular engineering, recombinant vaults can be functionally modified and targeted, and their contents can be controlled by packaging. Here, we review the development of engineered vaults as a platform for a wide variety of therapeutic applications and we examine future directions for this unique nanoparticle system.

Vaults are naturally-occurring ribonucleoprotein particles found in nearly all eukaryotic cells. They were named for their morphological resemblance to the vaulted ceilings of gothic cathedrals. These ubiquitous nanoparticles are quite abundant with 10(4)-10(6) copies found in the cytoplasm depending on cell type. The structural shell of the particle can self-assemble from 78 copies of a single protein, the major vault protein. This finding has allowed vaults to be bioengineered, resulting in a variety of new functions and capabilities directed toward overcoming many limitations posed by current gene and drug delivery systems. In this study, we demonstrate that recombinant vaults, with the addition of a cell penetration peptide, TAT, can be rapidly delivered to cells in vitro with significantly elevated binding and uptake efficiency. This TAT-vault nanoparticle could be a valuable tool for improving the retention and penetration of therapeutic drugs at tumor sites.

Vault nanoparticles were engineered to enhance their escape from the endosomal compartment by fusing a membrane lytic peptide derived from adenovirus protein VI (pVI) to the N-terminus of the major vault protein to form pVI-vaults. We demonstrate that these pVI-vaults disrupt the endosomal membrane using three different experimental protocols including (1) enhancement of DNA transfection, (2) co-delivery of a cytosolic ribotoxin, and (3) direct visualization by fluorescence. Furthermore, direct targeting of vaults to specific cell surface epidermal growth factor receptors led to enhanced cellular uptake and efficient delivery of vaults to the cytoplasm. This process was monitored with fluorescent vaults, and morphological changes in the endosomal compartment were observed. By combining targeting and endosomal escape into a single recombinant vault, high levels of transfection efficiency were achieved using low numbers of vault particles. These results demonstrate that engineered vaults are effective, efficient, and nontoxic nanoparticles for targeted delivery of biomaterials to the cell cytoplasm., (© 2011 American Chemical Society)

Vaults are naturally occurring ribonucleoprotein particles with an enormous interior volume, large enough to encapsulate hundreds of proteins. They are highly conserved and are present in nearly all eukaryotic cells ranging from 10(4) to 10(7) particles per cell. Recombinant vaults can be produced in vitro and engineered to allow cell targeting and protein packaging. These nanometer-sized particles have many desirable characteristics that may give them advantages for use as drug delivery vehicles. Using photoactivatable green fluorescent protein (PAGFP) labeled vaults, we demonstrate that the particles rapidly diffuse throughout the cytoplasm following single pixel photoactivation in live cells. Their in vivo movement remained relatively unchanged despite exposure to a variety of cellular stresses, suggesting that vaults are largely unconstrained in the cytoplasm. Fluorescence resonance energy transfer (FRET) was observed from polyethylene glycol (PEG) fused hybrid cells that expressed either CFP or YFP labeled vaults, indicating that vaults can exchange major vault protein (MVP) subunits in vivo. Investigation into the mechanism of this exchange in vitro using recombinant vaults demonstrated that they were capable of rapidly separating at the particle waist and reassembling back into whole vaults, supporting a half vault exchange mechanism. This data suggests a means whereby vaults can functionally interact with their cellular environment and deliver materials packaged within their interior.

Native vaults are nanoscale particles found abundantly in the cytoplasm of most eukaryotic cells. They have a capsule-like structure with a thin shell surrounding a "hollow" interior compartment. Recombinant vault particles were found to self-assemble following expression of the major vault protein (MVP) in a baculovirus expression system, and these particles are virtually identical to native vaults. Such particles have been recently studied as potential delivery vehicles. In this study, we focus on immobilization of vault particles on a solid substrate, such as glass, as a first step to study their interactions with cells. To this end, we first engineered the recombinant vaults by fusing two different tags to the C-terminus of MVP, a 3 amino acid RGD peptide and a 12 amino acid RGD-strep-tag peptide. We have demonstrated two strategies for immobilizing vaults on solid substrates. The barrel-and-cap structure of vault particles was observed for the first time, by atomic force microscopy (AFM), in a dry condition. This work proved the feasibility of immobilizing vault nanoparticles on a material surface, and the possibility of using vault nanoparticles as localized and sustainable drug carriers as well as a biocompatible surface moiety.

Vaults are large protein nanocapsules that may be useful as drug delivery vehicles due to their normal presence in humans, their large interior volume, their simple structural composition consisting of multiple copies of one protein, and a recombinant production system that also provides a means to tailor their structure. However, for vaults to be effective in such applications, efficient means to load the interiors of the capsules must be demonstrated. Here we describe the use of a domain derived from a vault lumen-associated protein as a carrier to target both gold nanoclusters and heterologous His-tagged proteins to specific binding sites on the vault interior wall.

Vaults, at 13 MDa, are the largest ribonucleoprotein particles known. In vitro, expression of the major vault protein (MVP) alone in Sf9 insect cells results in the production of recombinant particles with characteristic vault structure. With the ultimate goal of using recombinant vaults as nanocapsules for the delivery of biomolecules, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy, and light scattering) along with electron microscopy, to characterize the structural stability of vaults over a wide range of pH (3-8) and temperature (10-90 degrees C). Ten different conformational states of the vaults were identified over the pH and temperature range studied with the most stable region at pH 6-8 below 40 degrees C and least stable at pH 4-6 above 60 degrees C. A unique intermediate molten globulelike state was also identified at pH 6 and approximately 55 degrees C. EM imaging showed the opening of intact vaults into flowerlike structures when transitioning from neutral to acidic pH. This information has potential use in the development of recombinant vaults into nanocapsules for drug delivery since one mechanism by which therapeutic agents entrapped in vaults could be released is through an opening of the intact vault structure.

Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.

Vaults are 13 million Da ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies of a single protein, termed the major vault protein (MVP), is sufficient to form the minimal structure and entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, VPARP and TEP1, and a small untranslated vault RNA are also associated with vaults. We used the Sf9 insect cell expression system to form MVP-only recombinant vaults and performed a series of protein-mixing experiments to test whether this particle shell is able to exclude exogenous proteins from interacting with the vault interior. Surprisingly, we found that VPARP and TEP1 are able to incorporate into vaults even after the formation of the MVP vault particle shell is complete. Electrospray molecular mobility analysis and spectroscopic studies of vault-interacting proteins were used to confirm this result. Our results demonstrate that the protein shell of the recombinant vault particle is a dynamic structure and suggest a possible mechanism for in vivo assembly of vault-interacting proteins into preformed vaults. Finally, this study suggests that the vault interior may functionally interact with the cellular milieu.

Vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1) are components of the vault ribonucleoprotein complex. Vaults have been implicated in multidrug resistance of human tumors and are thought to be involved in macromolecular assembly and/or transport. Previous studies showed that VPARP-deficient mice were viable, fertile, and did not display any vault-related or telomerase-related phenotype, whereas disruption of telomerase-associated protein 1 in mice led to reduced stability of the vault RNA and affected its stable association with vaults, although there were no telomerase-related changes. In this study, we evaluated the susceptibility of Vparp-/- and Tep1-/- mice to dimethylhydrazine-induced colon tumorigenesis and urethane-induced lung tumorigenesis. Mice received i.p. injections of either 1 g/kg body weight of urethane twice a week for 2 weeks or 20 mg/kg body weight of dimethylhydrazine once a week for 10 weeks and were analyzed after 10 and 60 weeks, respectively. The colon tumor incidence and multiplicity were significantly higher and colon tumor latency was significantly shorter in Vparp-/- mice compared with wild-type mice. Increased colon tumor incidence, multiplicity, and reduced tumor latency were also seen in Tep1-/- mice, however, these results were statistically not significant. Lung tumor multiplicities were increased in both Vparp-/- and Tep1-/- mice but were not significant. The increase in carcinogen-induced tumors in VPARP-deficient mice is the only phenotype observed to date, and suggests a possible role for VPARP, directly or indirectly, in chemically induced neoplasia.

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.

The vault is a highly conserved ribonucleoprotein particle found in all higher eukaryotes. It has a barrel-shaped structure and is composed of the major vault protein (MVP); vault poly(ADP-ribose) polymerase (VPARP); telomerase-associated protein 1 (TEP1); and small untranslated RNA (vRNA). Although its strong conservation and high abundance indicate an important cellular role, the function of the vault is unknown. In humans, vaults have been implicated in multidrug resistance during chemotherapy. Recently, assembly of recombinant vaults has been established in insect cells expressing only MVP. Here, we demonstrate that co-expression of MVP with one or both of the other two vault proteins results in their co-assembly into regularly shaped vaults. Particles assembled from MVP with N-terminal peptide tags of various length are compared. Cryoelectron microscopy (cryoEM) and single-particle image reconstruction methods were used to determine the structure of nine recombinant vaults of various composition, as well as wild-type and TEP1-deficient mouse vaults. Recombinant vaults with MVP N-terminal peptide tags showed internal density that varied in size with the length of the tag. Reconstruction of a recombinant vault with a cysteine-rich tag revealed 48-fold rotational symmetry for the vault. A model is proposed for the organization of MVP within the vault with all of the MVP N termini interacting non-covalently at the vault midsection and 48 copies of MVP forming each half vault. CryoEM difference mapping localized VPARP to three density bands lining the inner surface of the vault. Difference maps designed to localize TEP1 showed only weak density inside of the caps, suggesting that TEP1 may interact with MVP via a small interaction region. In the absence of atomic-resolution structures for either VPARP or TEP1, fold recognition methods were applied. A total of 21 repeats were predicted for the TEP1 WD-repeat domain, suggesting an unusually large beta-propeller fold.

Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.

Multi-drug-resistant cancer cells frequently express elevated levels of ribonucleoprotein complexes termed vaults. The increased expression of vault proteins and their mRNAs has led to the suggestion that vaults may play a direct role in preventing drug toxicity. To further understand vault component up-regulation, the three proteins that comprise the vault, the major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein-1 (TEP1), were examined with respect to gene amplification and drug-induced chromatin remodeling. Gene amplification was not responsible for increased vault component levels in multi-drug-resistant cancer cell lines. The TATA-less murine MVP and human VPARP promoters were identified and functionally characterized. There was no significant activation of either the MVP or VPARP promoters in drug-resistant cell lines in comparison to their parental, drug-sensitive counterparts. Treatment of various cell lines with sodium butyrate, an inhibitor of histone deacetylase (HDAC), led to an increase in vault component protein levels. Furthermore, treatment with trichostatin A (TSA), a more specific inhibitor of HDAC, caused an increase in MVP protein, mRNA, and promoter activity. These results suggest that up-regulation of MVP in multi-drug resistance (MDR) may involve chromatin remodeling.

Vault RNA (vRNA) genes have been cloned from several vertebrates including rat, mouse, and humans. Their copy numbers vary, as does the length of the encoded RNA. We have determined that the mouse genome contains two vRNA genes; one is expressed the other is a pseudogene. In vitro transcription of the rat vRNA gene by RNA polymerase III has previously been shown to be dependent on a combination of both external and internal promoter sequence elements. By comparing the upstream regions of the vertebrate vRNA genes, a 25 bp conserved sequence and a TATA box can be identified. Furthermore, the unique arrangement of the internal promoter boxes (one A and two B boxes) is conserved in the expressed human vRNA genes even though a new RNA polymerase III termination sequence has evolved between the two B boxes.

Vaults are ribonucleoprotein particles with a distinct structure and a high degree of conservation between species. Although no function has been assigned to the complex yet, there is some evidence for a role of vaults in multidrug resistance. To confirm a direct relation between vaults and multidrug resistance, and to investigate other possible functions of vaults, we have generated a major vault protein (MVP/lung resistance-related protein) knockout mouse model. The MVP(-/-) mice are viable, healthy, and show no obvious abnormalities. We investigated the sensitivity of MVP(-/-) embryonic stem cells and bone marrow cells derived from the MVP-deficient mice to various cytostatic agents with different mechanisms of action. Neither the MVP(-/-) embryonic stem cells nor the MVP(-/-) bone marrow cells showed an increased sensitivity to any of the drugs examined, as compared with wild-type cells. Furthermore, the activities of the ABC-transporters P-glycoprotein, multidrug resistance-associated protein and breast cancer resistance protein were unaltered on MVP deletion in these cells. In addition, MVP wild-type and deficient mice were treated with the anthracycline doxorubicin. Both groups of mice responded similarly to the doxorubicin treatment. Our results suggest that MVP/vaults are not directly involved in the resistance to cytostatic agents.

Vaults are highly conserved ubiquitous ribonucleoprotein particles with an undefined function. Three protein species (p240/TEP1, p193/VPARP, and p100/MVP) and a small RNA comprise the 13-MDa vault particle. The expression of the unique 100-kDa major vault protein is sufficient to form the basic vault structure. Previously, we have shown that stable association of the vault RNA with the vault particle is dependent on its interaction with the p240/TEP1 protein. To identify other proteins that interact with the vault RNA, we used a UV-cross-linking assay. We find that a portion of the vault RNA is complexed with the La autoantigen in a separate smaller ribonucleoprotein particle. La interacts with the vault RNA (both in vivo and in vitro) presumably through binding to 3'-uridylates. Moreover, we also demonstrate that the La autoantigen is the 50-kDa protein that we have previously reported as a protein that co-purifies with vaults.