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Acid test for splicing
Acid test for splicing
Part 1 of the license CHONC, KHOO,
1. P. J., MAHBUBANI, H. M., C-Y. and BLOW, J. J. (1995) Purification of an MCM-containing complex as a component of the DNA replication licensing system Nature 375,41 B-421
MADINE, M. A., KHOO, C-Y., MILLS, A. D. and LASKEY, R. A. (1995) MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells Nature 375, 421-424 KUBOTA, Y., MIMURA, S., NISHIMOTO, S., TAKISAWA, H. and NOJIMA, H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor (1995) Cell 81, 601-609
The licensing-factor model was first proposed in 1988 as a mechanism by which eukaryotic cells ensure that DNA replication occurs once and only once per cell cycle. The basic tenet of the model was that breakdown of the nuclear envelope during mitosis allows access of licensing factor to the DNA, making it competent for replication. It was proposed that, during S phase, the process of replication inactivates licensing factor, preventing rereplication of the DNA. The race to identify licensing factor had commenced.. . The results described in these three papers have led to the identification of an MCM protein complex as a component of licensing factor. This complex contains three related proteins of 90-120 kDa, which have now been found in budding yeast [through studies of Mcrn (mini-chromosome maintenance) mutants], mammals, Drosophila and Xenopus. Chong et al. used biochemical fractionation of Xenopus egg extracts to identify the MCM complex, monitoring activity by complementation of an extract in which licensing factor had been inactivated by treatment with a
346
BORSI, The
alternative
L., BALZA, E., CACCERO, B., ALLEMANNI, G. and ZARDI, L. (1995) splicing pattern of tenascin-C pre-mRNA is controlled the extracellular pH /. Biol. Chem. 270,6243-6245
by
Alternative splicing is a mechanism whereby a single gene can generate multiple functionally different proteins. This mechanism has been described for a number of extracellular matrix proteins as well as for their corresponding receptors that belong to the integrin family. In some instances, alternative splicing has been shown to control the affinity of an integrin for its ligand, which in turn might regulate whether a cell can migrate or form stable matrix contacts. Alternative splicing is often found to be cell-type specific or developmentally regulated. There are two major splice variants of the glycoprotein tenascin-C (TN-C): tenascin-C large and tenascin-C small. One of these forms is incorporated into an extracellular matrix, whereas the other is not. The group of Zardi et al. has previously described an alternative splicing pattern of the matrix protein TN-C which correlates with the proliferation and post-mitotic states of cells. The same group has now found that relatively small changes in extracellular pH can dramatically alter the pattern of splicing without changing the total abundance of the two mRNAs for TN-C. In skin fibroblasts kept at pH 7.4, the 6 kb mRNA encoding the small splice variant constitutes 2% of total tenascin RNA, whereas at pH 6.7 it amounts to 98%. The authors speculate that changes in the pH of the microenvironment in certain normal organ systems, as well in certain pathological conditions, might change the splicing pattern. It will be important to determine the molecular details of how extracellular pH can affect the splicing machinery and whether this occurs not only in vitro but also under physiological conditions in the organism. It will also be interesting to determine to what extent pH-regulation controls alternative mRNA splicing.
protein kinase inhibitor. Kubota et al. showed that the MCM proteins bind to chromatin when incubated with a licensing competent (S phase) Xenopus egg extract but not with a licensing-incompetent GZ-like extract (M-phase extract treated with a protein kinase inhibitor). Madine et a/. found that immunodepletion of the MCM complex from Xenopus egg extracts by use of antibodies against Xenopus MCM3 removed licensing factor activity, which could be restored by readdition of MCM complex. However, there is clearly more to licensing factorthan just the proteins of the MCM complex. By performing biochemical fractionation, Chong et a/. identified a second TRENDS
component, RLF-B (for replication licensing-factor B), which is required in conjunction with the MCM complex to give licensing-factor activity. Although RLF-B has not been purified, the authors propose that its role may be to mediate attachment of MCM proteins to chromatin. Also, the nucleocytoplasmic localization of MCM proteins varies significantly between different cell types, so they are unlikely to be the only determinant of licensing-factor activity. Finally, there is considerable evidence, mainly from studies in yeast, that cyclin-cdc2 plays a role in preventing rereplication during G2 phase, so it will be interesting to learn how this complex is involved in the regulation of licensing factor.
IN CELL BIOLOGY
VOL.
5 SEPTEMBER
1995
Part 1 of the license CHONC, KHOO,
1. P. J., MAHBUBANI, H. M., C-Y. and BLOW, J. J. (1995) Purification of an MCM-containing complex as a component of the DNA replication licensing system Nature 375,41 B-421
MADINE, M. A., KHOO, C-Y., MILLS, A. D. and LASKEY, R. A. (1995) MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells Nature 375, 421-424 KUBOTA, Y., MIMURA, S., NISHIMOTO, S., TAKISAWA, H. and NOJIMA, H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor (1995) Cell 81, 601-609
The licensing-factor model was first proposed in 1988 as a mechanism by which eukaryotic cells ensure that DNA replication occurs once and only once per cell cycle. The basic tenet of the model was that breakdown of the nuclear envelope during mitosis allows access of licensing factor to the DNA, making it competent for replication. It was proposed that, during S phase, the process of replication inactivates licensing factor, preventing rereplication of the DNA. The race to identify licensing factor had commenced.. . The results described in these three papers have led to the identification of an MCM protein complex as a component of licensing factor. This complex contains three related proteins of 90-120 kDa, which have now been found in budding yeast [through studies of Mcrn (mini-chromosome maintenance) mutants], mammals, Drosophila and Xenopus. Chong et al. used biochemical fractionation of Xenopus egg extracts to identify the MCM complex, monitoring activity by complementation of an extract in which licensing factor had been inactivated by treatment with a
346
BORSI, The
alternative
L., BALZA, E., CACCERO, B., ALLEMANNI, G. and ZARDI, L. (1995) splicing pattern of tenascin-C pre-mRNA is controlled the extracellular pH /. Biol. Chem. 270,6243-6245
by
Alternative splicing is a mechanism whereby a single gene can generate multiple functionally different proteins. This mechanism has been described for a number of extracellular matrix proteins as well as for their corresponding receptors that belong to the integrin family. In some instances, alternative splicing has been shown to control the affinity of an integrin for its ligand, which in turn might regulate whether a cell can migrate or form stable matrix contacts. Alternative splicing is often found to be cell-type specific or developmentally regulated. There are two major splice variants of the glycoprotein tenascin-C (TN-C): tenascin-C large and tenascin-C small. One of these forms is incorporated into an extracellular matrix, whereas the other is not. The group of Zardi et al. has previously described an alternative splicing pattern of the matrix protein TN-C which correlates with the proliferation and post-mitotic states of cells. The same group has now found that relatively small changes in extracellular pH can dramatically alter the pattern of splicing without changing the total abundance of the two mRNAs for TN-C. In skin fibroblasts kept at pH 7.4, the 6 kb mRNA encoding the small splice variant constitutes 2% of total tenascin RNA, whereas at pH 6.7 it amounts to 98%. The authors speculate that changes in the pH of the microenvironment in certain normal organ systems, as well in certain pathological conditions, might change the splicing pattern. It will be important to determine the molecular details of how extracellular pH can affect the splicing machinery and whether this occurs not only in vitro but also under physiological conditions in the organism. It will also be interesting to determine to what extent pH-regulation controls alternative mRNA splicing.
protein kinase inhibitor. Kubota et al. showed that the MCM proteins bind to chromatin when incubated with a licensing competent (S phase) Xenopus egg extract but not with a licensing-incompetent GZ-like extract (M-phase extract treated with a protein kinase inhibitor). Madine et a/. found that immunodepletion of the MCM complex from Xenopus egg extracts by use of antibodies against Xenopus MCM3 removed licensing factor activity, which could be restored by readdition of MCM complex. However, there is clearly more to licensing factorthan just the proteins of the MCM complex. By performing biochemical fractionation, Chong et a/. identified a second TRENDS
component, RLF-B (for replication licensing-factor B), which is required in conjunction with the MCM complex to give licensing-factor activity. Although RLF-B has not been purified, the authors propose that its role may be to mediate attachment of MCM proteins to chromatin. Also, the nucleocytoplasmic localization of MCM proteins varies significantly between different cell types, so they are unlikely to be the only determinant of licensing-factor activity. Finally, there is considerable evidence, mainly from studies in yeast, that cyclin-cdc2 plays a role in preventing rereplication during G2 phase, so it will be interesting to learn how this complex is involved in the regulation of licensing factor.
IN CELL BIOLOGY
VOL.
5 SEPTEMBER
1995