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RNAi surges on: application to cultured mammalian cells
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News & Comment
TRENDS in Genetics Vol.17 No.8 August 2001
RNAi surges on: application to cultured mammalian cells The discovery that potent, sequence-specific inactivation of gene function can be induced by double-stranded RNA (dsRNA) has led to a revolution in reverse genetic analysis. The apparent ubiquity of the biochemical pathways involved means the approach, termed RNAi (for dsRNA-mediated interference has been demonstrated in various species of protozoa, hydra, flatworms, roundworms, arthropods and vertebrates, many not previously thought of as good genetic systems. For mammals, however, it was thought RNAi might be suitable only for studies on the oocyte and preimplantation embryo. In mammalian cells other than these, dsRNA triggers a nonspecific inhibition of protein synthesis that overwhelms any sequence-specific RNAi effect. Tuschl and colleagues1 have now demonstrated that the provision of an intermediate of the RNAi pathway can induce the sequence-specific effect without activating the general inhibition of translation in mammalian cells. Elbashir et al.1 showed that synthetic, 21-nucleotide RNAs, called small interfering RNAs (siRNAs), base paired such that they have
two-nucleotide 3’ overhangs, can specifically inhibit gene expression in cultured cells. Upon co-transfection with siRNAs, specific reductions in luciferase reporter gene expression occur in tissue cultured cells from human, monkey and mouse, as well as Drosophila melanogaster. In the mammalian cells, the effect was not as absolute as in the insect cells, with reductions in luciferase activity of up to 25-fold. Expression of one of the luciferase genes was not affected in one of the human cell lines examined. The study also examined endogenous genes. Again, the researchers used specific siRNAs, introduced into cultured human cells by liposome-mediated transfection, and measured the levels of the targeted endogenous proteins using antibodies. Although no reduction was observed in vimentin levels, a 90% reduction in lamin A/C protein was observed and lamin B1 and NuMA (nuclear mitotic apparatus protein) were also markedly reduced. Perhaps further developments, such as the use of multiple siRNAs aimed at a single gene, might improve the inhibition. The siRNAmediated RNAi effects might not be as
complete or as reliable, with regard to cell type or target gene, as other systems, but the applicability of RNAi in mammalian systems has been extended substantially by this work. The development of siRNAs is another step in the realization of the enormous potential of RNAi. The technique should be immediately applicable to reverse genetic analysis of the genes found in the human genome sequence. The pace of developments in the RNAi field gives the impression that any obstacle to a potential application of the technology will be overcome. If the spreading of the RNAi effect between cells throughout an organism could be obtained in mammals, in the same way as occurs in the nematode Caenorhabditis elegans, then RNAi could herald a new epoch in medicine. 1 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498
Ian A. Hope [email protected]
Cytoplasmic incompatibility and maternal-haploid Cytoplasmic incompatibility (CI) is an intriguing phenomenon, widespread in arthropods, that is induced by the maternally inherited endocellular bacteria Wolbachia. It can be described as an embryonic mortality occurring in crosses between infected males and uninfected females, whereas fertility is normal in the reverse cross, or if both partners are infected. The genes involved are unknown, but there is a general consensus that CI involves at least two bacterial functions: mod (for modification) and resc (for rescue). mod, the ‘poison’, is deposited on paternal chromosomes before Wolbachia are shed from maturing sperm, whereas resc, the ‘antidote’, is expressed by Wolbachia in infected eggs and saves the embryo from death. Embryonic mortality is due to the loss of paternal chromosomes, occurring at the first mitotic division. Chromosomes behave normally until metaphase, but paternal chromatids http://tig.trends.com
fail to separate in anaphase, resulting in the formation of a chromatin bridge in telophase. Loppin et al.1 recently provided an accurate cytological description of maternal-haploid (mh), a Drosophila melanogaster maternal-effect mutation. In fertilized eggs produced by mh homozygous mothers, paternal chromosomes are lost during the first mitosis or soon after, resulting in haploidy and embryo death. This observation demonstrates that paternal chromosomes are not functional after fertilization, unless maternal factors render them so. The authors note that the resemblance with CI is striking: in crosses involving mh homozygous mothers, paternal chromosomes behave exactly as in crosses where CI occurs (that is, in crosses between males infected by Wolbachia and uninfected females). What is the link between mh and CI? We see two hypotheses.
First, mh and mod might target the same sites on paternal chromosomes. Indeed, mod could occupy the target sites of mh, and thus disrupt the normal processing of anaphase. By interacting with mod, resc would allow the mh activity, and restore embryonic viability. Identifying the mh targets could then provide an indirect way to identify the mod function. Another, more speculative, hypothesis is that mh and resc are paralogous genes. In other words, the mod–resc system might be present in both the host and the Wolbachia genomes. Gene exchanges from the symbiont to the nucleus (or the other way round) could explain this pattern. Under this hypothesis, the mh phenotype would result from the same molecular process as CI: the inability of mh homozygous mothers to produce a ‘resc-like’ function and, thus, to save the embryo from the effect of a ‘mod-like’
0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Genetics Vol.17 No.8 August 2001
RNAi surges on: application to cultured mammalian cells The discovery that potent, sequence-specific inactivation of gene function can be induced by double-stranded RNA (dsRNA) has led to a revolution in reverse genetic analysis. The apparent ubiquity of the biochemical pathways involved means the approach, termed RNAi (for dsRNA-mediated interference has been demonstrated in various species of protozoa, hydra, flatworms, roundworms, arthropods and vertebrates, many not previously thought of as good genetic systems. For mammals, however, it was thought RNAi might be suitable only for studies on the oocyte and preimplantation embryo. In mammalian cells other than these, dsRNA triggers a nonspecific inhibition of protein synthesis that overwhelms any sequence-specific RNAi effect. Tuschl and colleagues1 have now demonstrated that the provision of an intermediate of the RNAi pathway can induce the sequence-specific effect without activating the general inhibition of translation in mammalian cells. Elbashir et al.1 showed that synthetic, 21-nucleotide RNAs, called small interfering RNAs (siRNAs), base paired such that they have
two-nucleotide 3’ overhangs, can specifically inhibit gene expression in cultured cells. Upon co-transfection with siRNAs, specific reductions in luciferase reporter gene expression occur in tissue cultured cells from human, monkey and mouse, as well as Drosophila melanogaster. In the mammalian cells, the effect was not as absolute as in the insect cells, with reductions in luciferase activity of up to 25-fold. Expression of one of the luciferase genes was not affected in one of the human cell lines examined. The study also examined endogenous genes. Again, the researchers used specific siRNAs, introduced into cultured human cells by liposome-mediated transfection, and measured the levels of the targeted endogenous proteins using antibodies. Although no reduction was observed in vimentin levels, a 90% reduction in lamin A/C protein was observed and lamin B1 and NuMA (nuclear mitotic apparatus protein) were also markedly reduced. Perhaps further developments, such as the use of multiple siRNAs aimed at a single gene, might improve the inhibition. The siRNAmediated RNAi effects might not be as
complete or as reliable, with regard to cell type or target gene, as other systems, but the applicability of RNAi in mammalian systems has been extended substantially by this work. The development of siRNAs is another step in the realization of the enormous potential of RNAi. The technique should be immediately applicable to reverse genetic analysis of the genes found in the human genome sequence. The pace of developments in the RNAi field gives the impression that any obstacle to a potential application of the technology will be overcome. If the spreading of the RNAi effect between cells throughout an organism could be obtained in mammals, in the same way as occurs in the nematode Caenorhabditis elegans, then RNAi could herald a new epoch in medicine. 1 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498
Ian A. Hope [email protected]
Cytoplasmic incompatibility and maternal-haploid Cytoplasmic incompatibility (CI) is an intriguing phenomenon, widespread in arthropods, that is induced by the maternally inherited endocellular bacteria Wolbachia. It can be described as an embryonic mortality occurring in crosses between infected males and uninfected females, whereas fertility is normal in the reverse cross, or if both partners are infected. The genes involved are unknown, but there is a general consensus that CI involves at least two bacterial functions: mod (for modification) and resc (for rescue). mod, the ‘poison’, is deposited on paternal chromosomes before Wolbachia are shed from maturing sperm, whereas resc, the ‘antidote’, is expressed by Wolbachia in infected eggs and saves the embryo from death. Embryonic mortality is due to the loss of paternal chromosomes, occurring at the first mitotic division. Chromosomes behave normally until metaphase, but paternal chromatids http://tig.trends.com
fail to separate in anaphase, resulting in the formation of a chromatin bridge in telophase. Loppin et al.1 recently provided an accurate cytological description of maternal-haploid (mh), a Drosophila melanogaster maternal-effect mutation. In fertilized eggs produced by mh homozygous mothers, paternal chromosomes are lost during the first mitosis or soon after, resulting in haploidy and embryo death. This observation demonstrates that paternal chromosomes are not functional after fertilization, unless maternal factors render them so. The authors note that the resemblance with CI is striking: in crosses involving mh homozygous mothers, paternal chromosomes behave exactly as in crosses where CI occurs (that is, in crosses between males infected by Wolbachia and uninfected females). What is the link between mh and CI? We see two hypotheses.
First, mh and mod might target the same sites on paternal chromosomes. Indeed, mod could occupy the target sites of mh, and thus disrupt the normal processing of anaphase. By interacting with mod, resc would allow the mh activity, and restore embryonic viability. Identifying the mh targets could then provide an indirect way to identify the mod function. Another, more speculative, hypothesis is that mh and resc are paralogous genes. In other words, the mod–resc system might be present in both the host and the Wolbachia genomes. Gene exchanges from the symbiont to the nucleus (or the other way round) could explain this pattern. Under this hypothesis, the mh phenotype would result from the same molecular process as CI: the inability of mh homozygous mothers to produce a ‘resc-like’ function and, thus, to save the embryo from the effect of a ‘mod-like’
0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.