The Experts below are selected from a list of 1746 Experts worldwide ranked by ideXlab platform
Leonard H. Rome - One of the best experts on this subject based on the ideXlab platform.
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Increased susceptibility of vault poly(ADP-ribose) polymerase-deficient mice to carcinogen-induced tumorigenesis.
Cancer Research, 2005Co-Authors: Sujna Raval-fernandes, Valerie A. Kickhoefer, Christina R. Kitchen, Leonard H. RomeAbstract: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.
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the p80 homology region of TEP1 is sufficient for its association with the telomerase and vault rnas and the vault particle
Nucleic Acids Research, 2005Co-Authors: Michael J Poderycki, Lea Harrington, Leonard H. Rome, Valerie A. KickhoeferAbstract: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.
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cryoelectron microscopy imaging of recombinant and tissue derived vaults localization of the mvp n termini and vparp
Journal of Molecular Biology, 2004Co-Authors: Yeshi Mikyas, Miriam Makabi, Sujna Ravalfernandes, Lea Harrington, Phoebe L. Stewart, Valerie A. Kickhoefer, Leonard H. RomeAbstract: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 β-propeller fold.
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Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.
Molecular and Cellular Biology, 2004Co-Authors: Bryan E Snow, Valerie A. Kickhoefer, Leonard H. Rome, Natalie Erdmann, Wen Zhou, Andrew Wakeham, Marla Gomez, Lea HarringtonAbstract:Telomerase is a ribonucleoprotein (RNP) complex that replenishes telomere loss due to incomplete DNA replication in almost all eukaryotes. A loss of telomerase activity leads to an attrition of telomeric DNA which, in turn, is known to trigger end-to-end chromosome fusions, genomic instability, and cell arrest or death (31). Telomerase expression is thus critical to the prolonged viability of several human cell types and in the majority of human cancers (3). Elucidating the molecular machinery that regulates telomerase activity and its ability to maintain telomere length is critical to an understanding of malignant transformation. Telomerase contains two core components, telomerase reverse transcriptase (TERT) and telomerase RNA, the latter serving as an integral template for the de novo synthesis of telomeric DNA. Both TERT and telomerase RNA are required for the reconstitution of telomerase activity in vitro (2, 75). In mice, the disruption of either component also abolishes telomerase activity, leading to telomere attrition, genetic instability, and eventual infertility (4, 15, 26, 44, 48, 61, 76). Several other telomerase-associated proteins have been identified in mammals (22); these include telomerase-associated protein 1 (TEP1), which binds telomerase RNA (23, 55) and which was cloned based on its homology to ciliate Tetrahymena thermophila protein p80 (8, 20, 23, 55). T. thermophila p80 was identified as a species copurifying with telomerase, although it subsequently was found unlikely to be a core telomerase component (8, 20, 49). Although deletion of the T. thermophila p80 gene resulted in slight telomere lengthening (51), no change in telomere length was observed upon breeding of mTEP1-deficient mice for up to seven generations. Telomerase activity was not affected in the absence of either the p80 gene or mTEP1 (47, 51). Disruption of mTEP1 did not affect the levels of telomerase RNA or its association with the telomerase RNP (34), and TEP1 appeared to associate with only a fraction of the total telomerase activity in immortalized cell extracts (Y. Liu and L. Harrington, unpublished data). Although these results suggest that TEP1 is nonessential for telomerase function in normal mouse tissues, the possibility of genetic redundancy with other telomerase-associated proteins cannot be excluded. Indeed, other telomerase-associated proteins have been identified and have been shown to interact with telomerase RNA in mammals; these include La, L22, Staufen, DKC, and several heterogeneous nuclear RNPs (11, 16-19, 29, 42, 43, 52). TEP1 is an integral component of another RNP, the vault particle. Mammalian vaults, the largest known mammalian RNPs (13 MDa), are composed of at least four components, major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), TEP1, and one or more small vault RNAs (vRNAs) (71, 73). Vaults possess a distinct morphology that is highly conserved. Purified vaults display a unique eightfold barrel-like symmetry structure with caps on each end (32, 38). Although the function of the vault particle has remained elusive, its highly conserved structure, its ubiquitous distribution, and its up-regulation in several human drug-resistant cancers have led to the speculation that vaults have an important cellular function and may be carriers involved in intracellular transport (54, 71, 73). The absence of TEP1 completely disrupts the stable association of vRNA with the purified vault particle and results in decreases in the levels and stability of vRNA (34). Therefore, TEP1 is an integral vault protein and is important for the stabilization and recruitment of vRNA to the vault particle (34). VPARP, the catalytic vault protein component, contains regions with similarity to BRCT, a poly(ADP-ribose) polymerase (PARP) catalytic domain, inter-α-trypsin and putative von Willebrand type A domains, and a C-terminal MVP-interacting domain (35, 71, 73). The putative VPARP catalytic domain shares 28% identity with the catalytic domain of PARP 1 (PARP1). Like that in PARP1, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction, and the substrates for this vault-associated PARP activity are MVP and VPARP itself (35). Thus, VPARP is a unique member of the PARP family. In addition to its association with vaults, VPARP has also been found at other cellular locations, such as the nucleus and mitotic spindle (35), indicating that it may possess multiple roles in vivo. Here, we report that VPARP interacts with TEP1 and associates with telomerase activity in cell extracts, suggesting that VPARP and TEP1 may play roles in both cytoplasmic and nuclear RNP complexes. We generated mice deficient in mVparp or both mTEP1 and mVparp and investigated telomerase function and vault structure in their absence.
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Up-regulation of vaults may be necessary but not sufficient for multidrug resistance.
International Journal of Cancer, 2001Co-Authors: Amara C. Siva, Valerie A. Kickhoefer, Andrew G Stephen, Sujna Raval-fernandes, Rik J. Scheper, Michael J. Lafemina, Leonard H. RomeAbstract:Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w. vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA). Increased levels of MVP, vault-associated vRNA and vaults have been linked directly to non-P-glycoprotein–mediated multidrug resistance (MDR). To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines. Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug-sensitive cells. Furthermore, protein analysis of subcellular fractions of the drug-sensitive, MVP-transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line. Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines. Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up-regulation alone is not sufficient to confer the drug-resistant phenotype. This implies a requirement of an additional factor(s) for vault-mediated MDR. © 2001 Wiley-Liss, Inc.
Valerie A. Kickhoefer - One of the best experts on this subject based on the ideXlab platform.
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Increased susceptibility of vault poly(ADP-ribose) polymerase-deficient mice to carcinogen-induced tumorigenesis.
Cancer Research, 2005Co-Authors: Sujna Raval-fernandes, Valerie A. Kickhoefer, Christina R. Kitchen, Leonard H. RomeAbstract: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.
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the p80 homology region of TEP1 is sufficient for its association with the telomerase and vault rnas and the vault particle
Nucleic Acids Research, 2005Co-Authors: Michael J Poderycki, Lea Harrington, Leonard H. Rome, Valerie A. KickhoeferAbstract: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.
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cryoelectron microscopy imaging of recombinant and tissue derived vaults localization of the mvp n termini and vparp
Journal of Molecular Biology, 2004Co-Authors: Yeshi Mikyas, Miriam Makabi, Sujna Ravalfernandes, Lea Harrington, Phoebe L. Stewart, Valerie A. Kickhoefer, Leonard H. RomeAbstract: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 β-propeller fold.
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Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.
Molecular and Cellular Biology, 2004Co-Authors: Bryan E Snow, Valerie A. Kickhoefer, Leonard H. Rome, Natalie Erdmann, Wen Zhou, Andrew Wakeham, Marla Gomez, Lea HarringtonAbstract:Telomerase is a ribonucleoprotein (RNP) complex that replenishes telomere loss due to incomplete DNA replication in almost all eukaryotes. A loss of telomerase activity leads to an attrition of telomeric DNA which, in turn, is known to trigger end-to-end chromosome fusions, genomic instability, and cell arrest or death (31). Telomerase expression is thus critical to the prolonged viability of several human cell types and in the majority of human cancers (3). Elucidating the molecular machinery that regulates telomerase activity and its ability to maintain telomere length is critical to an understanding of malignant transformation. Telomerase contains two core components, telomerase reverse transcriptase (TERT) and telomerase RNA, the latter serving as an integral template for the de novo synthesis of telomeric DNA. Both TERT and telomerase RNA are required for the reconstitution of telomerase activity in vitro (2, 75). In mice, the disruption of either component also abolishes telomerase activity, leading to telomere attrition, genetic instability, and eventual infertility (4, 15, 26, 44, 48, 61, 76). Several other telomerase-associated proteins have been identified in mammals (22); these include telomerase-associated protein 1 (TEP1), which binds telomerase RNA (23, 55) and which was cloned based on its homology to ciliate Tetrahymena thermophila protein p80 (8, 20, 23, 55). T. thermophila p80 was identified as a species copurifying with telomerase, although it subsequently was found unlikely to be a core telomerase component (8, 20, 49). Although deletion of the T. thermophila p80 gene resulted in slight telomere lengthening (51), no change in telomere length was observed upon breeding of mTEP1-deficient mice for up to seven generations. Telomerase activity was not affected in the absence of either the p80 gene or mTEP1 (47, 51). Disruption of mTEP1 did not affect the levels of telomerase RNA or its association with the telomerase RNP (34), and TEP1 appeared to associate with only a fraction of the total telomerase activity in immortalized cell extracts (Y. Liu and L. Harrington, unpublished data). Although these results suggest that TEP1 is nonessential for telomerase function in normal mouse tissues, the possibility of genetic redundancy with other telomerase-associated proteins cannot be excluded. Indeed, other telomerase-associated proteins have been identified and have been shown to interact with telomerase RNA in mammals; these include La, L22, Staufen, DKC, and several heterogeneous nuclear RNPs (11, 16-19, 29, 42, 43, 52). TEP1 is an integral component of another RNP, the vault particle. Mammalian vaults, the largest known mammalian RNPs (13 MDa), are composed of at least four components, major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), TEP1, and one or more small vault RNAs (vRNAs) (71, 73). Vaults possess a distinct morphology that is highly conserved. Purified vaults display a unique eightfold barrel-like symmetry structure with caps on each end (32, 38). Although the function of the vault particle has remained elusive, its highly conserved structure, its ubiquitous distribution, and its up-regulation in several human drug-resistant cancers have led to the speculation that vaults have an important cellular function and may be carriers involved in intracellular transport (54, 71, 73). The absence of TEP1 completely disrupts the stable association of vRNA with the purified vault particle and results in decreases in the levels and stability of vRNA (34). Therefore, TEP1 is an integral vault protein and is important for the stabilization and recruitment of vRNA to the vault particle (34). VPARP, the catalytic vault protein component, contains regions with similarity to BRCT, a poly(ADP-ribose) polymerase (PARP) catalytic domain, inter-α-trypsin and putative von Willebrand type A domains, and a C-terminal MVP-interacting domain (35, 71, 73). The putative VPARP catalytic domain shares 28% identity with the catalytic domain of PARP 1 (PARP1). Like that in PARP1, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction, and the substrates for this vault-associated PARP activity are MVP and VPARP itself (35). Thus, VPARP is a unique member of the PARP family. In addition to its association with vaults, VPARP has also been found at other cellular locations, such as the nucleus and mitotic spindle (35), indicating that it may possess multiple roles in vivo. Here, we report that VPARP interacts with TEP1 and associates with telomerase activity in cell extracts, suggesting that VPARP and TEP1 may play roles in both cytoplasmic and nuclear RNP complexes. We generated mice deficient in mVparp or both mTEP1 and mVparp and investigated telomerase function and vault structure in their absence.
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Up-regulation of vaults may be necessary but not sufficient for multidrug resistance.
International Journal of Cancer, 2001Co-Authors: Amara C. Siva, Valerie A. Kickhoefer, Andrew G Stephen, Sujna Raval-fernandes, Rik J. Scheper, Michael J. Lafemina, Leonard H. RomeAbstract:Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w. vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA). Increased levels of MVP, vault-associated vRNA and vaults have been linked directly to non-P-glycoprotein–mediated multidrug resistance (MDR). To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines. Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug-sensitive cells. Furthermore, protein analysis of subcellular fractions of the drug-sensitive, MVP-transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line. Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines. Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up-regulation alone is not sufficient to confer the drug-resistant phenotype. This implies a requirement of an additional factor(s) for vault-mediated MDR. © 2001 Wiley-Liss, Inc.
Phoebe L. Stewart - One of the best experts on this subject based on the ideXlab platform.
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cryoelectron microscopy imaging of recombinant and tissue derived vaults localization of the mvp n termini and vparp
Journal of Molecular Biology, 2004Co-Authors: Yeshi Mikyas, Miriam Makabi, Sujna Ravalfernandes, Lea Harrington, Phoebe L. Stewart, Valerie A. Kickhoefer, Leonard H. RomeAbstract: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 β-propeller fold.
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the telomerase vault associated protein TEP1 is required for vault rna stability and its association with the vault particle
Journal of Cell Biology, 2001Co-Authors: Valerie A. Kickhoefer, Lea Harrington, Phoebe L. Stewart, Lawrence B Kong, Bryan E Snow, Leonard H. RomeAbstract:Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTEP1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTEP1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTEP1−/− mice. Although vaults purified from the livers of mTEP1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTEP1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
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The Telomerase/Vault-Associated Protein TEP1 Is Required for Vault RNA Stability and Its Association with the Vault Particle
Journal of Cell Biology, 2001Co-Authors: Valerie A. Kickhoefer, Lea Harrington, Phoebe L. Stewart, Lawrence B Kong, Bryan E Snow, Leonard H. RomeAbstract:Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTEP1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTEP1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTEP1−/− mice. Although vaults purified from the livers of mTEP1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTEP1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
Lea Harrington - One of the best experts on this subject based on the ideXlab platform.
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the p80 homology region of TEP1 is sufficient for its association with the telomerase and vault rnas and the vault particle
Nucleic Acids Research, 2005Co-Authors: Michael J Poderycki, Lea Harrington, Leonard H. Rome, Valerie A. KickhoeferAbstract: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.
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cryoelectron microscopy imaging of recombinant and tissue derived vaults localization of the mvp n termini and vparp
Journal of Molecular Biology, 2004Co-Authors: Yeshi Mikyas, Miriam Makabi, Sujna Ravalfernandes, Lea Harrington, Phoebe L. Stewart, Valerie A. Kickhoefer, Leonard H. RomeAbstract: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 β-propeller fold.
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Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.
Molecular and Cellular Biology, 2004Co-Authors: Bryan E Snow, Valerie A. Kickhoefer, Leonard H. Rome, Natalie Erdmann, Wen Zhou, Andrew Wakeham, Marla Gomez, Lea HarringtonAbstract:Telomerase is a ribonucleoprotein (RNP) complex that replenishes telomere loss due to incomplete DNA replication in almost all eukaryotes. A loss of telomerase activity leads to an attrition of telomeric DNA which, in turn, is known to trigger end-to-end chromosome fusions, genomic instability, and cell arrest or death (31). Telomerase expression is thus critical to the prolonged viability of several human cell types and in the majority of human cancers (3). Elucidating the molecular machinery that regulates telomerase activity and its ability to maintain telomere length is critical to an understanding of malignant transformation. Telomerase contains two core components, telomerase reverse transcriptase (TERT) and telomerase RNA, the latter serving as an integral template for the de novo synthesis of telomeric DNA. Both TERT and telomerase RNA are required for the reconstitution of telomerase activity in vitro (2, 75). In mice, the disruption of either component also abolishes telomerase activity, leading to telomere attrition, genetic instability, and eventual infertility (4, 15, 26, 44, 48, 61, 76). Several other telomerase-associated proteins have been identified in mammals (22); these include telomerase-associated protein 1 (TEP1), which binds telomerase RNA (23, 55) and which was cloned based on its homology to ciliate Tetrahymena thermophila protein p80 (8, 20, 23, 55). T. thermophila p80 was identified as a species copurifying with telomerase, although it subsequently was found unlikely to be a core telomerase component (8, 20, 49). Although deletion of the T. thermophila p80 gene resulted in slight telomere lengthening (51), no change in telomere length was observed upon breeding of mTEP1-deficient mice for up to seven generations. Telomerase activity was not affected in the absence of either the p80 gene or mTEP1 (47, 51). Disruption of mTEP1 did not affect the levels of telomerase RNA or its association with the telomerase RNP (34), and TEP1 appeared to associate with only a fraction of the total telomerase activity in immortalized cell extracts (Y. Liu and L. Harrington, unpublished data). Although these results suggest that TEP1 is nonessential for telomerase function in normal mouse tissues, the possibility of genetic redundancy with other telomerase-associated proteins cannot be excluded. Indeed, other telomerase-associated proteins have been identified and have been shown to interact with telomerase RNA in mammals; these include La, L22, Staufen, DKC, and several heterogeneous nuclear RNPs (11, 16-19, 29, 42, 43, 52). TEP1 is an integral component of another RNP, the vault particle. Mammalian vaults, the largest known mammalian RNPs (13 MDa), are composed of at least four components, major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), TEP1, and one or more small vault RNAs (vRNAs) (71, 73). Vaults possess a distinct morphology that is highly conserved. Purified vaults display a unique eightfold barrel-like symmetry structure with caps on each end (32, 38). Although the function of the vault particle has remained elusive, its highly conserved structure, its ubiquitous distribution, and its up-regulation in several human drug-resistant cancers have led to the speculation that vaults have an important cellular function and may be carriers involved in intracellular transport (54, 71, 73). The absence of TEP1 completely disrupts the stable association of vRNA with the purified vault particle and results in decreases in the levels and stability of vRNA (34). Therefore, TEP1 is an integral vault protein and is important for the stabilization and recruitment of vRNA to the vault particle (34). VPARP, the catalytic vault protein component, contains regions with similarity to BRCT, a poly(ADP-ribose) polymerase (PARP) catalytic domain, inter-α-trypsin and putative von Willebrand type A domains, and a C-terminal MVP-interacting domain (35, 71, 73). The putative VPARP catalytic domain shares 28% identity with the catalytic domain of PARP 1 (PARP1). Like that in PARP1, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction, and the substrates for this vault-associated PARP activity are MVP and VPARP itself (35). Thus, VPARP is a unique member of the PARP family. In addition to its association with vaults, VPARP has also been found at other cellular locations, such as the nucleus and mitotic spindle (35), indicating that it may possess multiple roles in vivo. Here, we report that VPARP interacts with TEP1 and associates with telomerase activity in cell extracts, suggesting that VPARP and TEP1 may play roles in both cytoplasmic and nuclear RNP complexes. We generated mice deficient in mVparp or both mTEP1 and mVparp and investigated telomerase function and vault structure in their absence.
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the telomerase vault associated protein TEP1 is required for vault rna stability and its association with the vault particle
Journal of Cell Biology, 2001Co-Authors: Valerie A. Kickhoefer, Lea Harrington, Phoebe L. Stewart, Lawrence B Kong, Bryan E Snow, Leonard H. RomeAbstract:Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTEP1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTEP1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTEP1−/− mice. Although vaults purified from the livers of mTEP1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTEP1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
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The Telomerase/Vault-Associated Protein TEP1 Is Required for Vault RNA Stability and Its Association with the Vault Particle
Journal of Cell Biology, 2001Co-Authors: Valerie A. Kickhoefer, Lea Harrington, Phoebe L. Stewart, Lawrence B Kong, Bryan E Snow, Leonard H. RomeAbstract:Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTEP1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTEP1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTEP1−/− mice. Although vaults purified from the livers of mTEP1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTEP1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
Bryan E Snow - One of the best experts on this subject based on the ideXlab platform.
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Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.
Molecular and Cellular Biology, 2004Co-Authors: Bryan E Snow, Valerie A. Kickhoefer, Leonard H. Rome, Natalie Erdmann, Wen Zhou, Andrew Wakeham, Marla Gomez, Lea HarringtonAbstract:Telomerase is a ribonucleoprotein (RNP) complex that replenishes telomere loss due to incomplete DNA replication in almost all eukaryotes. A loss of telomerase activity leads to an attrition of telomeric DNA which, in turn, is known to trigger end-to-end chromosome fusions, genomic instability, and cell arrest or death (31). Telomerase expression is thus critical to the prolonged viability of several human cell types and in the majority of human cancers (3). Elucidating the molecular machinery that regulates telomerase activity and its ability to maintain telomere length is critical to an understanding of malignant transformation. Telomerase contains two core components, telomerase reverse transcriptase (TERT) and telomerase RNA, the latter serving as an integral template for the de novo synthesis of telomeric DNA. Both TERT and telomerase RNA are required for the reconstitution of telomerase activity in vitro (2, 75). In mice, the disruption of either component also abolishes telomerase activity, leading to telomere attrition, genetic instability, and eventual infertility (4, 15, 26, 44, 48, 61, 76). Several other telomerase-associated proteins have been identified in mammals (22); these include telomerase-associated protein 1 (TEP1), which binds telomerase RNA (23, 55) and which was cloned based on its homology to ciliate Tetrahymena thermophila protein p80 (8, 20, 23, 55). T. thermophila p80 was identified as a species copurifying with telomerase, although it subsequently was found unlikely to be a core telomerase component (8, 20, 49). Although deletion of the T. thermophila p80 gene resulted in slight telomere lengthening (51), no change in telomere length was observed upon breeding of mTEP1-deficient mice for up to seven generations. Telomerase activity was not affected in the absence of either the p80 gene or mTEP1 (47, 51). Disruption of mTEP1 did not affect the levels of telomerase RNA or its association with the telomerase RNP (34), and TEP1 appeared to associate with only a fraction of the total telomerase activity in immortalized cell extracts (Y. Liu and L. Harrington, unpublished data). Although these results suggest that TEP1 is nonessential for telomerase function in normal mouse tissues, the possibility of genetic redundancy with other telomerase-associated proteins cannot be excluded. Indeed, other telomerase-associated proteins have been identified and have been shown to interact with telomerase RNA in mammals; these include La, L22, Staufen, DKC, and several heterogeneous nuclear RNPs (11, 16-19, 29, 42, 43, 52). TEP1 is an integral component of another RNP, the vault particle. Mammalian vaults, the largest known mammalian RNPs (13 MDa), are composed of at least four components, major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), TEP1, and one or more small vault RNAs (vRNAs) (71, 73). Vaults possess a distinct morphology that is highly conserved. Purified vaults display a unique eightfold barrel-like symmetry structure with caps on each end (32, 38). Although the function of the vault particle has remained elusive, its highly conserved structure, its ubiquitous distribution, and its up-regulation in several human drug-resistant cancers have led to the speculation that vaults have an important cellular function and may be carriers involved in intracellular transport (54, 71, 73). The absence of TEP1 completely disrupts the stable association of vRNA with the purified vault particle and results in decreases in the levels and stability of vRNA (34). Therefore, TEP1 is an integral vault protein and is important for the stabilization and recruitment of vRNA to the vault particle (34). VPARP, the catalytic vault protein component, contains regions with similarity to BRCT, a poly(ADP-ribose) polymerase (PARP) catalytic domain, inter-α-trypsin and putative von Willebrand type A domains, and a C-terminal MVP-interacting domain (35, 71, 73). The putative VPARP catalytic domain shares 28% identity with the catalytic domain of PARP 1 (PARP1). Like that in PARP1, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction, and the substrates for this vault-associated PARP activity are MVP and VPARP itself (35). Thus, VPARP is a unique member of the PARP family. In addition to its association with vaults, VPARP has also been found at other cellular locations, such as the nucleus and mitotic spindle (35), indicating that it may possess multiple roles in vivo. Here, we report that VPARP interacts with TEP1 and associates with telomerase activity in cell extracts, suggesting that VPARP and TEP1 may play roles in both cytoplasmic and nuclear RNP complexes. We generated mice deficient in mVparp or both mTEP1 and mVparp and investigated telomerase function and vault structure in their absence.
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the telomerase vault associated protein TEP1 is required for vault rna stability and its association with the vault particle
Journal of Cell Biology, 2001Co-Authors: Valerie A. Kickhoefer, Lea Harrington, Phoebe L. Stewart, Lawrence B Kong, Bryan E Snow, Leonard H. RomeAbstract:Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTEP1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTEP1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTEP1−/− mice. Although vaults purified from the livers of mTEP1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTEP1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
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The Telomerase/Vault-Associated Protein TEP1 Is Required for Vault RNA Stability and Its Association with the Vault Particle
Journal of Cell Biology, 2001Co-Authors: Valerie A. Kickhoefer, Lea Harrington, Phoebe L. Stewart, Lawrence B Kong, Bryan E Snow, Leonard H. RomeAbstract:Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTEP1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTEP1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTEP1−/− mice. Although vaults purified from the livers of mTEP1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTEP1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
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telomerase associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo
Molecular and Cellular Biology, 2000Co-Authors: Bryan E Snow, Valerie A. Kickhoefer, Andrew Wakeham, Prakash M Hande, Gabriela M Baerlocher, David Yeung, Annick Itie, David P Siderovski, Peter M LansdorpAbstract:Most eukaryotic chromosome ends are maintained by a ribonucleoprotein (RNP) complex called telomerase. Telomerase is a reverse transcriptase that uses an integral RNA component to catalyze the addition of telomeric repeats to the 3′ end of single-stranded telomeric DNA (8). In many organisms, the telomerase complex is a large (750- to 1,000-kDa) RNP containing an integral RNA, a reverse transcriptase protein subunit, and several associated proteins. The telomerase RNA component provides a template for telomere DNA synthesis, and its essential role in telomerase activity, telomere length maintenance, and chromosome stability has been demonstrated in ciliates, yeast, and mice (2, 14–16, 29, 39, 42). The telomerase reverse transcriptase (TERT) was first identified in the yeasts Saccharomyces cerevisiae (EST2) and Schizosaccharomyces pombe (trt1+) and the ciliate Euplotes aediculatus (p123) (32, 36) and subsequently in humans (hTERT) (12, 25, 34, 36, 38). Mutations of conserved amino acids within the reverse transcriptase domain of S. cerevisiae Est2 and in human TERT result in the loss of telomerase activity (6, 12, 32, 38, 46). In rabbit reticulocyte lysates, human telomerase activity is reconstituted by the addition of human TERT (hTERT) and the telomerase RNA (1, 46). In addition to the presumed core telomerase components, consisting of the telomerase RNA and TERT, several proteins associated with telomerase activity have also been identified. In humans, the “foldosome” proteins hsp90 and p23 and three telomerase RNA binding proteins, dyskerin, L22, and hStau, are each associated with telomerase activity in cell extracts (18, 28, 35). In S. cerevisiae, the Sm protein is necessary for the stability of the yeast telomerase RNA (44), while three other proteins, Est1, Est3, and Cdc13, are dispensable for telomerase activity but are required for telomere length maintenance (30, 33, 45). In the ciliate Tetrahymena thermophila, two proteins that copurify with telomerase, p80 and p95 (4, 7, 10), bind to telomerase RNA and telomeric DNA, although their precise role in the Tetrahymena telomerase complex is not yet clear (4, 7). The mammalian homolog of p80, TEP1, is associated with telomerase activity in human, mouse, and rat immortalized-cell extracts (11, 37). The amino-terminal 900 amino acids of TEP1, which contain the region homologous to Tetrahymena p80, also interacted with telomerase RNA in an in vivo RNA-protein interaction assay (11). Despite its association with telomerase components, the role of TEP1 in telomerase function is unknown. Recently, TEP1 has also been identified as a component of a large cytoplasmic RNP termed the vault particle (24). The genetic characterization of these proteins is critical to our understanding of the complexity, composition, and regulation of telomerase in vivo. We utilized homologous recombination to disrupt the first mammalian telomerase-associated protein to be identified, mTEP1, in mice and embryonic stem (ES) cells and analyzed the effects on telomerase activity and telomere length maintenance.
