【研究进展】Science：法国科学家发现人类肝RNA沉默机制
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Human RNA silences viral DNA
MicroRNA plays an unexpected role in the process, researchers report in Science | By Charles Q Choi
RNA silencing can defend against viruses in humans, French scientists report in this week's Science. Surprisingly, say the scientists, microRNA (miRNA) appears to form the basis of this system.
"MiRNAs were thought to be involved in the regulation of endogenous genes, whereas exogenous RNAs, in particular viral RNAs, were thought to be regulated by siRNA [small interfering RNA]," lead author Charles-Henri Lecellier at the Institute of Plant Molecular Biology in Strasbourg, France, told The Scientist.
Prior studies have revealed that RNA interference can destroy viruses in plants and insects, but a similar role in vertebrates has not been demonstrated. Since RNA silencing can suppress endogenous retroviruses from mobilizing in plants, yeast, worms, and flies, Lecellier and colleagues reasoned that retrotransposition of mammalian exogenous viruses might also prove vulnerable. They chose as their model system the primate foamy virus type 1, a retrovirus akin to HIV.
PFV-1 accumulation in cultured human embryonic kidney cells was strongly enhanced by the expression of the P19 silencing suppressor, suggesting that a siRNA or miRNA pathway limited PFV-1 replication in human cells, because P19 specifically binds to and inactivates both.
To identify the target and means of human RNA silencing, the investigators fused viral sequences spanning the PFV-1 genome to a green fluorescent protein (GFP)–tagged reporter gene into constructs cotransfected with PFV-1 into baby hamster kidney cells. Northern and Western analysis revealed GFP levels from construct F11 were disproportionately reduced compared to F11 mRNA accumulation, which reminded researchers of miRNA translational inhibition. The DIANA-microT algorithm revealed a high probability match between the F11 sequence and the human miR-32.
Further studies demonstrated miR-32 silencing was suppressed in P19-expressing cells. Also, anti-miR-32 locked nucleic acid oligonucleotide almost doubled progeny virus production, unlike anti-miR-23, suggesting miR-32 has a direct, sequence-specific antiviral effect.
In plants and insects, all viruses targeted by RNA interference encode proteins that suppress RNA silencing. Further studies found that in PFV-1, Tas, a viral transactivator, was that protein.
In Arabidopsis, transgenic Tas expression strongly decreased siRNAs and led to developmental anomalies reminiscent of those elicited by suppressors interfering with miRNA functions, such as leaf elongation and serration, suggesting Tas suppresses a fundamental step conserved from plants to mammals shared between the miRNA and siRNA pathways.
Like Tas, another protein, AC2, encoded by the DNA plant viruses, geminiviruses, is a viral transactivator that can suppress RNA silencing. "We want to investigate whether transactivation and suppression are linked or completely separate," Lecellier said.
Researchers currently think each cell type harbors its own specific miRNA repertoire, Lecellier said. "This idea could partially explain some of the differences in viral permissivity observed between specific tissues," he said, with viruses preferentially replicating in cell types where antiviral miRNAs are not expressed or are only weakly expressed.
A miRNA response could also lead to the emergence of viral quasispecies, as viruses that can rapidly introduce synonymous mutations into their genomes, such as HIV or influenza, do so to evade silencing by miRNA. "The emergence of quasispecies is important for resistance to antiviral strategies, so studying this miRNA response could be important for studying resistance," Lecellier said.
The team saw no evidence that human cells used siRNAs to disable viruses. "So it'd be interesting to investigate whether or not mammals have lost the ability to respond to viruses using siRNAs because they have a more advanced immune system than plants and flies and worms," said Phillip Zamore at the University of Massachusetts, who did not participate in this study. It was uncertain whether the miRNAs were part of a dedicated antiviral response or whether they accidentally silenced viral RNA, he said. "It'd be interesting to see whether expression of miRNAs are vastly upregulated in viral infection," he told The Scientist.
Future directions should involve testing any of the several hundred human microRNAs against viruses such as HIV and influenza, said Shou-Wei Ding at the University of California at Riverside, who did not participate in this study. "Also, they studied this in cell culture, and it'd be interesting to look at this at the whole animal level," Ding told The Scientist.
Links for this article
C. Lecellier et al., "A cellular microRNA mediates antiviral defense in human cells," Science, 308:557-60. April 22, 2005.
http://www.sciencemag.org
Charles-Henri Lecellier
http://www.sigu7.jussieu.fr/B2M/pages/doc/2K4/virord11va.html
A.J. Hamilton, D.C. Baulcombe, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science, 286:950-2, October 29, 1999.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/10542148/]
H. Li et al. "Induction and suppression of RNA silencing by an animal virus," Science, 296:1319-21, May 17, 2002.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/12016316http://www.biomedcentral.com/pubmed/12016316]
C. Lecellier, A. Saib. "Foamy viruses: between retroviruses and pararetroviruses," Virology, 271:1-8, May 25, 2000.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/10814564http://www.biomedcentral.com/pubmed/10814564]
Phillip Zamore
http://www.umassmed.edu/bmp/faculty/zamore.cfm
Shou-Wei Ding
http://www.cepceb.ucr.edu/members/ding.htm
据《科学》杂志4月23日报道，MicroRNA在人体发育过程中起着意想不到的作用。
法国科学家称核糖核酸沉默机制（RNA silencing）能够抵抗人体中的病毒，不可思议的是，小RNA (miRNA)竟然是组成这一机制的基础。
“科学家一直认为miRNA与调节内源基因有关，而小分子干扰核糖核酸（iRNA）能够控制外源基因，尤其是病毒RNA基因，”该文的主要作者、来自法国斯特拉斯堡的植物分子生物学院的查利斯·亨利·莱西利亚在《科学》杂志上说道。
科学家们先前的研究显示，核糖核酸干扰（RNAi）能够消灭植物以及昆虫体内的病毒，但目前还没有研究表明它在脊椎动物也拥有类似功能。自从科学家们发现RNA沉默机制能够压抑内生反转录病毒，使其无法在植物、酵母、蚯蚓以及苍蝇等等体内游动，莱西利亚和他的同事就推断哺乳动物体内的外源病毒基因的反转录移位功能应当也是同样脆弱。因此他们选择了一种原始的1型泡沫病毒（PFV-1）——一种与人体免疫缺损病毒（HIV）同类的逆转录酶病毒——作为实验的模拟系统。
由于P19沉默干扰基因的表达，PFV-1病毒在人工“人体胚胎肾脏细胞株”（human embryonic kidney cells）中聚集并且变得十分明显。这意味着，由于P19细胞控制着siRNA和miRNA，而且这两种核糖核酸的路径也控制着PFV-1在人体细胞中的繁殖。
为了确定人体RNA沉默机制的具体作用，研究者们将PFV-1病毒所含有的病毒基因序列与一种附有报告基因绿色荧光蛋白（GFP）链接在一起，然后使它们与人体胚胎肾脏细胞一起染上病毒。报告显示，F11架构中的绿色荧光蛋白与F11mRNA聚集中所含有的绿色荧光蛋白比例完全不同。这一点使研究者们想到了miRNA转录抑制。利用DIANA-microT运算法则计算后发现F11序列与人体miR-32具有高度的相似性。
进一步的研究表明，miR-32沉默受到P19表达细胞的压抑。同样，受抗miR-32细胞控制的核酸低聚核苷酸使子代病毒的繁殖量增加了一倍，并不是像抗miR-32细胞所起的作用一样。这意味着miR-32起着直接反病毒作用。
植物与昆虫中，所有的核糖核酸干扰（RNAi）能够消灭的病毒都含有可以抑制核糖核酸沉默的蛋白质。而在后来的研究结果显示，PFV-1中含有一种叫做Tas的病毒反激活蛋白。
一种叫做“阿拉伯芥”的植物中，转基因Tas能够大量减少siRNAs，并且使干扰基因引发的细胞产生发育变异现象，同时干扰miRNA的功能，例如植物叶子变长、长出锯齿等。这意味着Tas具有抑制miRNA 和siRNA某种基本功能的作用，而这种功能是植物与哺乳动物共同拥有的。
类似Tas的另一种蛋白质，AC2，其中含有植物基因病毒，也是一种病毒反激活蛋白，同样能够压抑RNA沉默机制。
目前，研究者们认为每种类型的细胞都有自己独有的miRNA细胞库。莱西利亚说：“这样的概念能够部分解答一些有关病毒反应方面的问题。”他认为，在细胞种类中，优先繁殖的病毒是处在抗病毒miRNAs没有表现的地方，或者表现并不明显的地方。
MiRNA的反应也会导致基因准种群出现，如果病毒能够快速使其基因组发生变异，例如HIV与流感病毒，它就能避免受到miRNA沉默机制的影响。“基因准种群的出现对于躲避抗病毒功能的影响是十分重要的，因此研究这种miRNA反应也十分重要，” 莱西利亚说。
这一研究小组并没有发现人类细胞能够利用siRNA来消灭病毒。“所以研究哺乳动物是用还具有利用siRNA来对付病毒的能力是一个十分有趣的课题，因为与蚯蚓、苍蝇这些昆虫相比，哺乳动物拥有更加先进的免疫系统，”麻省理工大学的飞利浦·赛摩说。他并没有参加这次研究活动。虽然miRNAs是否是抗病毒反应的原因之一，或者只是偶然对病毒核糖核酸起到了沉默作用，但是他认为：“不论miRNAs的表达是否会抑制病毒传染，这项研究都会引起科学界的广泛关注。”
美国加州大学的丁守维（音译）称，未来的研究方向可能会包括测试几百个人体microRNAs对诸如HIV病毒和流感病毒等病毒的反应，他也未参加此次研究。他说：“他们已经在细胞培养室做了试验，如果在动物身上做相关实验的话会引起科学界更大的关注。”
法国研究人员发现，哺乳动物细胞能关闭入侵的病毒。当病毒感染细胞后，它把自己的基因插入细胞的基因组，这样在细胞复制时也产生许多的病毒拷贝。研究人员已经知道植物和昆虫用RNA干扰使病毒基因沉默，在这个过程中，小的RNA分子将自己插入到基因表达机器中使某个基因沉默。动物也将RNA干扰用在一个调节功能上：它们在发育过程中通过RNA干扰改变自己基因的表达。Charles Henri Lecellier和同事现在发现，人类细胞也用RNA干扰来阻碍一个侵袭哺乳类的病毒的积累。
MicroRNA plays an unexpected role in the process, researchers report in Science | By Charles Q Choi
RNA silencing can defend against viruses in humans, French scientists report in this week's Science. Surprisingly, say the scientists, microRNA (miRNA) appears to form the basis of this system.
"MiRNAs were thought to be involved in the regulation of endogenous genes, whereas exogenous RNAs, in particular viral RNAs, were thought to be regulated by siRNA [small interfering RNA]," lead author Charles-Henri Lecellier at the Institute of Plant Molecular Biology in Strasbourg, France, told The Scientist.
Prior studies have revealed that RNA interference can destroy viruses in plants and insects, but a similar role in vertebrates has not been demonstrated. Since RNA silencing can suppress endogenous retroviruses from mobilizing in plants, yeast, worms, and flies, Lecellier and colleagues reasoned that retrotransposition of mammalian exogenous viruses might also prove vulnerable. They chose as their model system the primate foamy virus type 1, a retrovirus akin to HIV.
PFV-1 accumulation in cultured human embryonic kidney cells was strongly enhanced by the expression of the P19 silencing suppressor, suggesting that a siRNA or miRNA pathway limited PFV-1 replication in human cells, because P19 specifically binds to and inactivates both.
To identify the target and means of human RNA silencing, the investigators fused viral sequences spanning the PFV-1 genome to a green fluorescent protein (GFP)–tagged reporter gene into constructs cotransfected with PFV-1 into baby hamster kidney cells. Northern and Western analysis revealed GFP levels from construct F11 were disproportionately reduced compared to F11 mRNA accumulation, which reminded researchers of miRNA translational inhibition. The DIANA-microT algorithm revealed a high probability match between the F11 sequence and the human miR-32.
Further studies demonstrated miR-32 silencing was suppressed in P19-expressing cells. Also, anti-miR-32 locked nucleic acid oligonucleotide almost doubled progeny virus production, unlike anti-miR-23, suggesting miR-32 has a direct, sequence-specific antiviral effect.
In plants and insects, all viruses targeted by RNA interference encode proteins that suppress RNA silencing. Further studies found that in PFV-1, Tas, a viral transactivator, was that protein.
In Arabidopsis, transgenic Tas expression strongly decreased siRNAs and led to developmental anomalies reminiscent of those elicited by suppressors interfering with miRNA functions, such as leaf elongation and serration, suggesting Tas suppresses a fundamental step conserved from plants to mammals shared between the miRNA and siRNA pathways.
Like Tas, another protein, AC2, encoded by the DNA plant viruses, geminiviruses, is a viral transactivator that can suppress RNA silencing. "We want to investigate whether transactivation and suppression are linked or completely separate," Lecellier said.
Researchers currently think each cell type harbors its own specific miRNA repertoire, Lecellier said. "This idea could partially explain some of the differences in viral permissivity observed between specific tissues," he said, with viruses preferentially replicating in cell types where antiviral miRNAs are not expressed or are only weakly expressed.
A miRNA response could also lead to the emergence of viral quasispecies, as viruses that can rapidly introduce synonymous mutations into their genomes, such as HIV or influenza, do so to evade silencing by miRNA. "The emergence of quasispecies is important for resistance to antiviral strategies, so studying this miRNA response could be important for studying resistance," Lecellier said.
The team saw no evidence that human cells used siRNAs to disable viruses. "So it'd be interesting to investigate whether or not mammals have lost the ability to respond to viruses using siRNAs because they have a more advanced immune system than plants and flies and worms," said Phillip Zamore at the University of Massachusetts, who did not participate in this study. It was uncertain whether the miRNAs were part of a dedicated antiviral response or whether they accidentally silenced viral RNA, he said. "It'd be interesting to see whether expression of miRNAs are vastly upregulated in viral infection," he told The Scientist.
Future directions should involve testing any of the several hundred human microRNAs against viruses such as HIV and influenza, said Shou-Wei Ding at the University of California at Riverside, who did not participate in this study. "Also, they studied this in cell culture, and it'd be interesting to look at this at the whole animal level," Ding told The Scientist.
Links for this article
C. Lecellier et al., "A cellular microRNA mediates antiviral defense in human cells," Science, 308:557-60. April 22, 2005.
http://www.sciencemag.org
Charles-Henri Lecellier
http://www.sigu7.jussieu.fr/B2M/pages/doc/2K4/virord11va.html
A.J. Hamilton, D.C. Baulcombe, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science, 286:950-2, October 29, 1999.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/10542148/]
H. Li et al. "Induction and suppression of RNA silencing by an animal virus," Science, 296:1319-21, May 17, 2002.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/12016316http://www.biomedcentral.com/pubmed/12016316]
C. Lecellier, A. Saib. "Foamy viruses: between retroviruses and pararetroviruses," Virology, 271:1-8, May 25, 2000.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/10814564http://www.biomedcentral.com/pubmed/10814564]
Phillip Zamore
http://www.umassmed.edu/bmp/faculty/zamore.cfm
Shou-Wei Ding
http://www.cepceb.ucr.edu/members/ding.htm
据《科学》杂志4月23日报道，MicroRNA在人体发育过程中起着意想不到的作用。
法国科学家称核糖核酸沉默机制（RNA silencing）能够抵抗人体中的病毒，不可思议的是，小RNA (miRNA)竟然是组成这一机制的基础。
“科学家一直认为miRNA与调节内源基因有关，而小分子干扰核糖核酸（iRNA）能够控制外源基因，尤其是病毒RNA基因，”该文的主要作者、来自法国斯特拉斯堡的植物分子生物学院的查利斯·亨利·莱西利亚在《科学》杂志上说道。
科学家们先前的研究显示，核糖核酸干扰（RNAi）能够消灭植物以及昆虫体内的病毒，但目前还没有研究表明它在脊椎动物也拥有类似功能。自从科学家们发现RNA沉默机制能够压抑内生反转录病毒，使其无法在植物、酵母、蚯蚓以及苍蝇等等体内游动，莱西利亚和他的同事就推断哺乳动物体内的外源病毒基因的反转录移位功能应当也是同样脆弱。因此他们选择了一种原始的1型泡沫病毒（PFV-1）——一种与人体免疫缺损病毒（HIV）同类的逆转录酶病毒——作为实验的模拟系统。
由于P19沉默干扰基因的表达，PFV-1病毒在人工“人体胚胎肾脏细胞株”（human embryonic kidney cells）中聚集并且变得十分明显。这意味着，由于P19细胞控制着siRNA和miRNA，而且这两种核糖核酸的路径也控制着PFV-1在人体细胞中的繁殖。
为了确定人体RNA沉默机制的具体作用，研究者们将PFV-1病毒所含有的病毒基因序列与一种附有报告基因绿色荧光蛋白（GFP）链接在一起，然后使它们与人体胚胎肾脏细胞一起染上病毒。报告显示，F11架构中的绿色荧光蛋白与F11mRNA聚集中所含有的绿色荧光蛋白比例完全不同。这一点使研究者们想到了miRNA转录抑制。利用DIANA-microT运算法则计算后发现F11序列与人体miR-32具有高度的相似性。
进一步的研究表明，miR-32沉默受到P19表达细胞的压抑。同样，受抗miR-32细胞控制的核酸低聚核苷酸使子代病毒的繁殖量增加了一倍，并不是像抗miR-32细胞所起的作用一样。这意味着miR-32起着直接反病毒作用。
植物与昆虫中，所有的核糖核酸干扰（RNAi）能够消灭的病毒都含有可以抑制核糖核酸沉默的蛋白质。而在后来的研究结果显示，PFV-1中含有一种叫做Tas的病毒反激活蛋白。
一种叫做“阿拉伯芥”的植物中，转基因Tas能够大量减少siRNAs，并且使干扰基因引发的细胞产生发育变异现象，同时干扰miRNA的功能，例如植物叶子变长、长出锯齿等。这意味着Tas具有抑制miRNA 和siRNA某种基本功能的作用，而这种功能是植物与哺乳动物共同拥有的。
类似Tas的另一种蛋白质，AC2，其中含有植物基因病毒，也是一种病毒反激活蛋白，同样能够压抑RNA沉默机制。
目前，研究者们认为每种类型的细胞都有自己独有的miRNA细胞库。莱西利亚说：“这样的概念能够部分解答一些有关病毒反应方面的问题。”他认为，在细胞种类中，优先繁殖的病毒是处在抗病毒miRNAs没有表现的地方，或者表现并不明显的地方。
MiRNA的反应也会导致基因准种群出现，如果病毒能够快速使其基因组发生变异，例如HIV与流感病毒，它就能避免受到miRNA沉默机制的影响。“基因准种群的出现对于躲避抗病毒功能的影响是十分重要的，因此研究这种miRNA反应也十分重要，” 莱西利亚说。
这一研究小组并没有发现人类细胞能够利用siRNA来消灭病毒。“所以研究哺乳动物是用还具有利用siRNA来对付病毒的能力是一个十分有趣的课题，因为与蚯蚓、苍蝇这些昆虫相比，哺乳动物拥有更加先进的免疫系统，”麻省理工大学的飞利浦·赛摩说。他并没有参加这次研究活动。虽然miRNAs是否是抗病毒反应的原因之一，或者只是偶然对病毒核糖核酸起到了沉默作用，但是他认为：“不论miRNAs的表达是否会抑制病毒传染，这项研究都会引起科学界的广泛关注。”
美国加州大学的丁守维（音译）称，未来的研究方向可能会包括测试几百个人体microRNAs对诸如HIV病毒和流感病毒等病毒的反应，他也未参加此次研究。他说：“他们已经在细胞培养室做了试验，如果在动物身上做相关实验的话会引起科学界更大的关注。”
法国研究人员发现，哺乳动物细胞能关闭入侵的病毒。当病毒感染细胞后，它把自己的基因插入细胞的基因组，这样在细胞复制时也产生许多的病毒拷贝。研究人员已经知道植物和昆虫用RNA干扰使病毒基因沉默，在这个过程中，小的RNA分子将自己插入到基因表达机器中使某个基因沉默。动物也将RNA干扰用在一个调节功能上：它们在发育过程中通过RNA干扰改变自己基因的表达。Charles Henri Lecellier和同事现在发现，人类细胞也用RNA干扰来阻碍一个侵袭哺乳类的病毒的积累。
