The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Erik A.c. Wiemer - One of the best experts on this subject based on the ideXlab platform.
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Vaults and drug resistance: What we learn from an MVP/LRP knockout mouse model.
Cancer Research, 2004Co-Authors: Erik A.c. Wiemer, Marieke H. Mossink, George L. Scheffer, Rik J Scheper, Pieter SonneveldAbstract:2455 Vaults are cytoplasmic ribonucleoprotein particles of which a small fraction is associated with the nucleus. The vault complex consists of small Untranslated RNA molecules (vRNAs) of 88-141 bases and multiple copies of three proteins; the Mr 100.000 major vault protein (MVP) - or lung resistance-related protein (LRP) - the Mr 193.000 vault poly-(ADP-ribose) polymerase (vPARP) and the Mr 240.000 telomerase associated protein (TEP1). The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was proposed that vaults contribute to drug resistance by mediating the transport and/or sequestration of cytotoxic drugs. Supporting this hypothesis are reports showing that vaults are involved in the efflux of anthracyclines from the nucleus. In order to understand the physiological function of vaults and to examine its putative role in drug resistance we generated an MVP knockout mouse model (Cancer Res. 62, 7298-7304, 2002). Biochemical fractionation and electron microscopical analyses revealed that vault particles were absent from MVP−/− tissues. Immunoblot analysis indicated that the TEP1 and vPARP protein levels are severely decreased in MVP−/− tissues. The levels and half-life of the vRNA were also found to be reduced probably due to the diminished amounts of TEP1. Northern blot analysis showed that TEP1 and vPARP mRNA levels and polysome formation are unaffected suggesting that the presence of MVP is essential for TEP1 and vPARP protein stability. In order to study how drugs are handled in the presence and absence of vaults we examined the influx and efflux kinetics and subcellular distribution of the fluorescent drug daunorubicin in mouse embryonic fibroblasts (MEFs) derived from wild-type and MVP−/− littermates. MEFs were cultured in the presence of daunorubicin after which the cells were allowed to efflux the drug. The intracellular daunorubicin levels were monitored by FACS analysis. No difference in efflux kinetics was observed between wild-type and MVP−/− MEFs. Likewise the intracellular distribution of daunorubicin was similar in the two cell lines, with daunorubicin accumulating in the nucleus first, after which it was redistributed to cytoplasmic vesicular structures. Expression of a GFP-tagged MVP in MVP−/− cells resulted in the formation of vault particles and the concomitant stabilization of vPARP, TEP1 and vRNA, but did not lead to an increased daunorubicin efflux or to alterations in its intracellular distribution. Our data suggest that vaults are not directly involved in the extrusion of daunorubicin from cells or nuclei, nor do they function in the sequestration of the drug in cytoplasmic vesicles. These results confirm our initial findings that MVP−/− mice, and bone marrow cells derived from these mice, do not display hypersensitivity to cytotoxic drugs. We conclude that vaults do not seem to play a direct role in drug resistance.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP
Journal of Cell Science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21°C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21°C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.
Journal of cell science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.
Journal of cell science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The genomic sequence of the murine major vault protein and its promoter.
Gene, 2002Co-Authors: Marieke H. Mossink, Arend Van Zon, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Erna Fränzel-luiten, Erik A.c. WiemerAbstract:Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small Untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first Untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5′-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression.
Marieke H. Mossink - One of the best experts on this subject based on the ideXlab platform.
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Vaults and drug resistance: What we learn from an MVP/LRP knockout mouse model.
Cancer Research, 2004Co-Authors: Erik A.c. Wiemer, Marieke H. Mossink, George L. Scheffer, Rik J Scheper, Pieter SonneveldAbstract:2455 Vaults are cytoplasmic ribonucleoprotein particles of which a small fraction is associated with the nucleus. The vault complex consists of small Untranslated RNA molecules (vRNAs) of 88-141 bases and multiple copies of three proteins; the Mr 100.000 major vault protein (MVP) - or lung resistance-related protein (LRP) - the Mr 193.000 vault poly-(ADP-ribose) polymerase (vPARP) and the Mr 240.000 telomerase associated protein (TEP1). The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was proposed that vaults contribute to drug resistance by mediating the transport and/or sequestration of cytotoxic drugs. Supporting this hypothesis are reports showing that vaults are involved in the efflux of anthracyclines from the nucleus. In order to understand the physiological function of vaults and to examine its putative role in drug resistance we generated an MVP knockout mouse model (Cancer Res. 62, 7298-7304, 2002). Biochemical fractionation and electron microscopical analyses revealed that vault particles were absent from MVP−/− tissues. Immunoblot analysis indicated that the TEP1 and vPARP protein levels are severely decreased in MVP−/− tissues. The levels and half-life of the vRNA were also found to be reduced probably due to the diminished amounts of TEP1. Northern blot analysis showed that TEP1 and vPARP mRNA levels and polysome formation are unaffected suggesting that the presence of MVP is essential for TEP1 and vPARP protein stability. In order to study how drugs are handled in the presence and absence of vaults we examined the influx and efflux kinetics and subcellular distribution of the fluorescent drug daunorubicin in mouse embryonic fibroblasts (MEFs) derived from wild-type and MVP−/− littermates. MEFs were cultured in the presence of daunorubicin after which the cells were allowed to efflux the drug. The intracellular daunorubicin levels were monitored by FACS analysis. No difference in efflux kinetics was observed between wild-type and MVP−/− MEFs. Likewise the intracellular distribution of daunorubicin was similar in the two cell lines, with daunorubicin accumulating in the nucleus first, after which it was redistributed to cytoplasmic vesicular structures. Expression of a GFP-tagged MVP in MVP−/− cells resulted in the formation of vault particles and the concomitant stabilization of vPARP, TEP1 and vRNA, but did not lead to an increased daunorubicin efflux or to alterations in its intracellular distribution. Our data suggest that vaults are not directly involved in the extrusion of daunorubicin from cells or nuclei, nor do they function in the sequestration of the drug in cytoplasmic vesicles. These results confirm our initial findings that MVP−/− mice, and bone marrow cells derived from these mice, do not display hypersensitivity to cytotoxic drugs. We conclude that vaults do not seem to play a direct role in drug resistance.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP
Journal of Cell Science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21°C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21°C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.
Journal of cell science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.
Journal of cell science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The genomic sequence of the murine major vault protein and its promoter.
Gene, 2002Co-Authors: Marieke H. Mossink, Arend Van Zon, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Erna Fränzel-luiten, Erik A.c. WiemerAbstract:Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small Untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first Untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5′-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression.
Sabine Brantl - One of the best experts on this subject based on the ideXlab platform.
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The small Untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism
Molecular microbiology, 2006Co-Authors: Nadja Heidrich, Alberto Chinali, Ulf Gerth, Sabine BrantlAbstract:Summary Whereas about 70 small non-coding RNAs have been found in the Escherichia coli genome, relatively little is known about regulatory RNAs from Gram-positive bacteria. Here, we demonstrate that the recently iden- tified small Untranslated RNA SR1 from the Bacillus subtilis genome is a regulatory RNA involved in fine- tuning of arginine catabolism. 2D protein gel electro- phoresis indicated three possible SR1 targets that are regulated by the transcriptional activator AhrC, which was shown to be the primary target of SR1. In vitro pairing studies and an in vivo reporter gene test dem- onstrated a specific interaction between SR1 and ahrC mRNA. This interaction did not lead to degrada- tion of ahrC mRNA, but inhibited translation at a post- initiation stage. Our data show that the Hfq chaperone was not required for the stabilization of SR1 in vivo. The amount of SR1 was increased upon addition of L-arginine and L-ornithine, but not L-citrulline or L-proline.
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Implication of CcpN in the regulation of a novel Untranslated RNA (SR1) in Bacillus subtilis.
Molecular microbiology, 2005Co-Authors: Andreas Licht, Sven Preis, Sabine BrantlAbstract:Summary Antisense-RNAs have been investigated in detail over the past 20 years as the principal regulators in acces- sory DNA elements such as plasmids, phages and transposons. However, only a few examples of chro- mosomally encoded bacterial antisense RNAs were known. Meanwhile, a a a 70 small non-coding RNAs from the Escherichia coli genome have been found, the functions of the majority of which remain to be eluci- dated. Only one systematic search has been per- formed for Gram-positive bacteria, so far. Here, we report the identification of a novel small (205 nt) non- translated RNA - SR1 - encoded in the Bacillus sub- tilis genome. SR1 was predicted by a computational approach and verified by Northern blotting. Knockout or overexpression of SR1 did not affect growth. SR1 was derepressed under conditions of gluconeogene- sis, but repressed under glycolytic conditions. Two regulatory levels could be identified, one involving CcpA, the second, more important, involving the recently identified regulator CcpN.
Pieter Sonneveld - One of the best experts on this subject based on the ideXlab platform.
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Vaults and drug resistance: What we learn from an MVP/LRP knockout mouse model.
Cancer Research, 2004Co-Authors: Erik A.c. Wiemer, Marieke H. Mossink, George L. Scheffer, Rik J Scheper, Pieter SonneveldAbstract:2455 Vaults are cytoplasmic ribonucleoprotein particles of which a small fraction is associated with the nucleus. The vault complex consists of small Untranslated RNA molecules (vRNAs) of 88-141 bases and multiple copies of three proteins; the Mr 100.000 major vault protein (MVP) - or lung resistance-related protein (LRP) - the Mr 193.000 vault poly-(ADP-ribose) polymerase (vPARP) and the Mr 240.000 telomerase associated protein (TEP1). The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was proposed that vaults contribute to drug resistance by mediating the transport and/or sequestration of cytotoxic drugs. Supporting this hypothesis are reports showing that vaults are involved in the efflux of anthracyclines from the nucleus. In order to understand the physiological function of vaults and to examine its putative role in drug resistance we generated an MVP knockout mouse model (Cancer Res. 62, 7298-7304, 2002). Biochemical fractionation and electron microscopical analyses revealed that vault particles were absent from MVP−/− tissues. Immunoblot analysis indicated that the TEP1 and vPARP protein levels are severely decreased in MVP−/− tissues. The levels and half-life of the vRNA were also found to be reduced probably due to the diminished amounts of TEP1. Northern blot analysis showed that TEP1 and vPARP mRNA levels and polysome formation are unaffected suggesting that the presence of MVP is essential for TEP1 and vPARP protein stability. In order to study how drugs are handled in the presence and absence of vaults we examined the influx and efflux kinetics and subcellular distribution of the fluorescent drug daunorubicin in mouse embryonic fibroblasts (MEFs) derived from wild-type and MVP−/− littermates. MEFs were cultured in the presence of daunorubicin after which the cells were allowed to efflux the drug. The intracellular daunorubicin levels were monitored by FACS analysis. No difference in efflux kinetics was observed between wild-type and MVP−/− MEFs. Likewise the intracellular distribution of daunorubicin was similar in the two cell lines, with daunorubicin accumulating in the nucleus first, after which it was redistributed to cytoplasmic vesicular structures. Expression of a GFP-tagged MVP in MVP−/− cells resulted in the formation of vault particles and the concomitant stabilization of vPARP, TEP1 and vRNA, but did not lead to an increased daunorubicin efflux or to alterations in its intracellular distribution. Our data suggest that vaults are not directly involved in the extrusion of daunorubicin from cells or nuclei, nor do they function in the sequestration of the drug in cytoplasmic vesicles. These results confirm our initial findings that MVP−/− mice, and bone marrow cells derived from these mice, do not display hypersensitivity to cytotoxic drugs. We conclude that vaults do not seem to play a direct role in drug resistance.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP
Journal of Cell Science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21°C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21°C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.
Journal of cell science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.
Journal of cell science, 2003Co-Authors: Marieke H. Mossink, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Adriaan B Houtsmuller, Erik A.c. WiemerAbstract:Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small Untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.
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The genomic sequence of the murine major vault protein and its promoter.
Gene, 2002Co-Authors: Marieke H. Mossink, Arend Van Zon, George L. Scheffer, Pieter Sonneveld, Martijn Schoester, Rik J Scheper, Erna Fränzel-luiten, Erik A.c. WiemerAbstract:Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small Untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first Untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5′-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression.
Christopher M. Waters - One of the best experts on this subject based on the ideXlab platform.
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The Vc2 Cyclic di-GMP-Dependent Riboswitch of Vibrio cholerae Regulates Expression of an Upstream Putative Small RNA by Controlling RNA Stability
Journal of bacteriology, 2019Co-Authors: Benjamin R. Pursley, Nicolas L. Fernandez, Geoffrey B. Severin, Christopher M. WatersAbstract:ABSTRACT Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream noncoding RNAs with 3′ ends located immediately downstream of the Vc2 riboswitch. Our results suggest these putative small RNAs (sRNAs) are not generated by transcriptional termination but rather by preventing degradation of the upstream Untranslated RNA when c-di-GMP is bound to Vc2. IMPORTANCE Riboswitches are typically RNA elements located in the 5′ Untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or translation initiation. In this work, we report a novel mechanism of riboswitch-mediated gene regulation in Vibrio cholerae whereby a 3′ riboswitch, named Vc2, controls the stability of upstream Untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed putative sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structured RNA, we hypothesize this mechanism of gene regulation is widespread.
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The Vc2 cyclic di-GMP dependent riboswitch of Vibrio cholerae regulates expression of an upstream small RNA by controlling RNA stability
2019Co-Authors: Benjamin R. Pursley, Christopher M. WatersAbstract:SUMMARY Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off-switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream non-coding small RNAs (sRNA) with 3’-ends located immediately downstream of the Vc2 riboswitch. Our results suggest these sRNAs are not generated by transcriptional termination but rather by preventing degradation of the upstream Untranslated RNA when c-di-GMP is bound to Vc2. IMPORTANCE Riboswitches are typically RNA elements located in the 5’ Untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or the translation initiation. In this work, we report a novel mechanism of riboswitch mediated gene regulation in Vibrio cholerae whereby a 3’ riboswitch, named Vc2, controls the stability of upstream Untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structure RNA, we hypothesize this mechanism of gene regulation could be widespread.
