纯化的Argonaute2和siRNA形成了重组的人源RISC复合物
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Field: Biology & Biochemistry
Article Title: Purified Argonaute2 and an siRNA form recombinant human RISC
Authors: Rivas, FV;Tolia, NH;Song, JJ;Aragon, JP;Liu, JD;Hannon, GJ;Joshua-Tor, L
Journal: NAT STRUCT MOL BIOL
Volume: 12
Issue: 4
Page: 340-349
Q：Why do you think your paper is highly cited?
A：This paper showed definitively that human Argonaute2 (hAgo2) is indeed "Slicer," the catalytic component of the RISC complex. Moreover, it showed that all that is needed for slicer activity is Argonaute and the siRNA guide. Slicing, or mRNA endonucleolytic cleavage, is the key even in RNAi posttranscriptional silencing. This minimal system also provided the opportunity to examine what features of RISC activity reside in Argonaute and what resides elsewhere, perhaps in another component of RISC.
Q： Does it describe a new discovery or a new methodology that's useful to others?
A：This paper describes an expression system for hAgo2 in E. coli. It appears to be useful to others since we have been sending out this expression system to labs all over ever since the paper was published. This now can be used to examine other features of RISC, since one would be able to constitute various activities in vitro using this system as the starting point.
Q： Could you summarize the significance of your paper in layman's terms?
A：RNA interference (RNAi) has emerged as a widespread biological regulatory mechanism, a powerful tool for both basic and applied research, and as a therapeutic strategy of enormous potential. It has taken experimental biology by storm. In organisms from fungi and flies to plants and humans, RNAi plays an essential, multifaceted role in controlling gene expression. One of the best-studied RNAi mechanisms is the quashing of gene expression through the cleavage and destruction of templates for protein synthesis called "messenger RNA," or mRNA.
Until recently, however, the identity of the molecular scissors that actually cut messenger RNA during RNAi has remained elusive. The solution to this puzzle emerged when we showed that the structure of an Argonaute protein revealed a region that is the spitting image of another enzyme called Ribonuclease H (RNase H) that is known to cut RNA. This led to the proposal that Argonaute functions as the important "Slicer" in a molecular machine called RISC, which was known to carry out this critical step of RNA interference. We could also suggest how this would be guided by the siRNA to cut the mRNA.
In collaboration with my colleague Greg Hannon, we tested this in the mammalian cells and showed, based on the structure for human cells, Argonaute 2 was Slicer. However, this was shown for proteins that were purified directly from human cells. As there appeared to be a marked difference between the different human Argonautes, there was some possibility that a minor impurity was modulating the activity that we saw.
We therefore decided that we must produce the protein in cells that lack RNAi all together. The bacterium E. coli was the prime candidate for this. Being able to show that a protein produced in E. coli had this activity was the first important step, but this was closely followed by the realization that this provided a paired-down system that was optimal to test many known?or as yet unknown?discovered features of RNAi slicing activity, some of which were explored in this work.
Q： How did you become involved in this research, and were any problems encountered along the way?
A：RNAi is an exciting new field and, as the components of this important biological pathway were being unraveled, much of the work was done here at Cold Spring Harbor Lab. It made an enormous impact on biology in a very short period of time. What impressed me most was how much it benefited from an incredible marriage of genetics, biochemistry, molecular biology, and bioinformatics. We thought that there was a great opportunity for structural biology which could drive much of the biochemical and mechanistic studies. Indeed, our structural studies of Argonaute drove much of the biochemical work described in this paper.
Leemor Joshua-Tor, Ph. D.
Professor
Keck Structural Biology Laboratory
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY, USA
这篇访问Leemor教授的文章发表在今年六月份的Essential science indicators网站上。访问的主题是关于Leemor教授2004年发表在nature structural 上的一篇关于RNAi机制的文章。这一篇文章发表后在这个领域产生了巨大的影响。文章的题目是：Purified Argonaute2 and an siRNA form recombinant human RISC（纯化的Argonaute2和siRNA形成了重组的人源RISC复合物）。
几个名词的解释：
RISC（RNA—induced silencing complex）是一个在RNA干涉现象中起着关键作用的分子复合物，它和特定的小RNA片断结合后可以切割特定的信使RNA。
Slicer在词典中的意思是切薄片的人或者切片机。对于RNA的切割本文用了几个不同的词，有slice，cleavage，cut。如果是将RNA切成等分的一段一段就用slice，而如果只是在固定的点切割用的是cleavage，cut用得比较少一般就是泛指切割。
Q：你认为你的文章被大量引用的原因是什么？
A：这篇文章准确无误的显示人源 Argonaute 2（hAgo2）就是“切片机”——RISC复合物中的催化组分。而且这篇文章还证明了只要有Argonaute和 siRNA指导就会有切片活性。RNA的切片或者mRNA的核内剪切都是RNAi 翻译后抑制中的关键事件。通过该微型系统，也可判断哪些RISC的活性特点应该归于Argonaute，哪些可能是RISC的另一部分。
Q：那么这是一个新的发现还是一个对他人有用的新方法？
A：这篇文章描述了hAgo2在大肠杆菌中的表达系统。自文章发表以后，我们已经送出了很多这样的表达系统给世界各地的实验室，它看起来很管用。而以这个系统为起点可以在体外构建RISC的各种活性，这样就可以来研究RISC的其他特性。
Q：你能用通俗的语言总结一下你的文章的意义吗？
A：RNA 干扰（RNAi）是一种分布广泛的生物调节机制，也是一个对基础和应用研究都很强大的工具，同时它还是一种有着巨大潜力的治疗手段。它已经在实验生物学领域中掀起了风暴。生物中从真菌和苍蝇到植物和人类，RNAi都在控制基因表达中扮演了多重的关键角色。RNAi机制中研究的最彻底的就是通过剪切损毁作为蛋白合成模板的“信使RNA”来抑制基因表达。
然而直到现在到底是哪个分子剪刀在RNA干扰的过程中剪切信使RNA仍然不清楚。不过我们的研究使得这个问题的解决成为可能。我们提供了Argonaute蛋白的结构，这个结构的一段区域是另外一个叫做RNA水解酶H（RNase H）——它可以切割RNA——的分离图形。这个发现提示Argonaute可能在这个被称为RISC的分子机器中担任十分重要的“切片机”的功能。这个发现还可以提示siRNA是怎么样导引RISC切割mRNA的。
通过和我的同事Greg Hannon合作，我们在哺乳动物细胞中测试了这个机制。结果显示在人源细胞的结构中，Argonaute 2是“切片机”。但该结果是通过直接从人源细胞中纯化的蛋白所获得。我们发现不同人源Argonautes所进行的实验有显著差异，这可能是由于蛋白有少许不纯导致我们看到的活性不同。
因此我们决定必须在完全缺少RNAi系统的细胞中生产Argonaute 2，大肠杆菌是首选。证明大肠杆菌产生的蛋白质有活性是非常重要的第一步，然后我们很快认识到这提供了一个很适合测试许多已知或者未知的RNAi剪切活性特性的paired-down系统。我们自己在这项研究中也测试了一些。
Q：你是怎么开始这项研究的？在这个过程中你遇到困难了吗？
A：作为一个正在被揭开其面纱的的重要生物途径，RNAi 是一个让人兴奋的新领域。而很多这方面的工作都在冷船港的实验室完成。这些研究在短时间内就对生物学产生了巨大冲击。而让我印象最深刻的是这些研究从遗传学、生物化学、分子生物学和生物信息学结合中获得的成果。因此我们认为对结构生物学来说这也是一个伟大的机会，因为结构生物学往往可以推动很多生物化学及其机制的研究。事实上我们在这篇文章中关于Argonaute结构的研究也确实推动了这方面的生物化学工作。
编辑：西门吹血
Article Title: Purified Argonaute2 and an siRNA form recombinant human RISC
Authors: Rivas, FV;Tolia, NH;Song, JJ;Aragon, JP;Liu, JD;Hannon, GJ;Joshua-Tor, L
Journal: NAT STRUCT MOL BIOL
Volume: 12
Issue: 4
Page: 340-349
Q：Why do you think your paper is highly cited?
A：This paper showed definitively that human Argonaute2 (hAgo2) is indeed "Slicer," the catalytic component of the RISC complex. Moreover, it showed that all that is needed for slicer activity is Argonaute and the siRNA guide. Slicing, or mRNA endonucleolytic cleavage, is the key even in RNAi posttranscriptional silencing. This minimal system also provided the opportunity to examine what features of RISC activity reside in Argonaute and what resides elsewhere, perhaps in another component of RISC.
Q： Does it describe a new discovery or a new methodology that's useful to others?
A：This paper describes an expression system for hAgo2 in E. coli. It appears to be useful to others since we have been sending out this expression system to labs all over ever since the paper was published. This now can be used to examine other features of RISC, since one would be able to constitute various activities in vitro using this system as the starting point.
Q： Could you summarize the significance of your paper in layman's terms?
A：RNA interference (RNAi) has emerged as a widespread biological regulatory mechanism, a powerful tool for both basic and applied research, and as a therapeutic strategy of enormous potential. It has taken experimental biology by storm. In organisms from fungi and flies to plants and humans, RNAi plays an essential, multifaceted role in controlling gene expression. One of the best-studied RNAi mechanisms is the quashing of gene expression through the cleavage and destruction of templates for protein synthesis called "messenger RNA," or mRNA.
Until recently, however, the identity of the molecular scissors that actually cut messenger RNA during RNAi has remained elusive. The solution to this puzzle emerged when we showed that the structure of an Argonaute protein revealed a region that is the spitting image of another enzyme called Ribonuclease H (RNase H) that is known to cut RNA. This led to the proposal that Argonaute functions as the important "Slicer" in a molecular machine called RISC, which was known to carry out this critical step of RNA interference. We could also suggest how this would be guided by the siRNA to cut the mRNA.
In collaboration with my colleague Greg Hannon, we tested this in the mammalian cells and showed, based on the structure for human cells, Argonaute 2 was Slicer. However, this was shown for proteins that were purified directly from human cells. As there appeared to be a marked difference between the different human Argonautes, there was some possibility that a minor impurity was modulating the activity that we saw.
We therefore decided that we must produce the protein in cells that lack RNAi all together. The bacterium E. coli was the prime candidate for this. Being able to show that a protein produced in E. coli had this activity was the first important step, but this was closely followed by the realization that this provided a paired-down system that was optimal to test many known?or as yet unknown?discovered features of RNAi slicing activity, some of which were explored in this work.
Q： How did you become involved in this research, and were any problems encountered along the way?
A：RNAi is an exciting new field and, as the components of this important biological pathway were being unraveled, much of the work was done here at Cold Spring Harbor Lab. It made an enormous impact on biology in a very short period of time. What impressed me most was how much it benefited from an incredible marriage of genetics, biochemistry, molecular biology, and bioinformatics. We thought that there was a great opportunity for structural biology which could drive much of the biochemical and mechanistic studies. Indeed, our structural studies of Argonaute drove much of the biochemical work described in this paper.
Leemor Joshua-Tor, Ph. D.
Professor
Keck Structural Biology Laboratory
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY, USA
这篇访问Leemor教授的文章发表在今年六月份的Essential science indicators网站上。访问的主题是关于Leemor教授2004年发表在nature structural 上的一篇关于RNAi机制的文章。这一篇文章发表后在这个领域产生了巨大的影响。文章的题目是：Purified Argonaute2 and an siRNA form recombinant human RISC（纯化的Argonaute2和siRNA形成了重组的人源RISC复合物）。
几个名词的解释：
RISC（RNA—induced silencing complex）是一个在RNA干涉现象中起着关键作用的分子复合物，它和特定的小RNA片断结合后可以切割特定的信使RNA。
Slicer在词典中的意思是切薄片的人或者切片机。对于RNA的切割本文用了几个不同的词，有slice，cleavage，cut。如果是将RNA切成等分的一段一段就用slice，而如果只是在固定的点切割用的是cleavage，cut用得比较少一般就是泛指切割。
Q：你认为你的文章被大量引用的原因是什么？
A：这篇文章准确无误的显示人源 Argonaute 2（hAgo2）就是“切片机”——RISC复合物中的催化组分。而且这篇文章还证明了只要有Argonaute和 siRNA指导就会有切片活性。RNA的切片或者mRNA的核内剪切都是RNAi 翻译后抑制中的关键事件。通过该微型系统，也可判断哪些RISC的活性特点应该归于Argonaute，哪些可能是RISC的另一部分。
Q：那么这是一个新的发现还是一个对他人有用的新方法？
A：这篇文章描述了hAgo2在大肠杆菌中的表达系统。自文章发表以后，我们已经送出了很多这样的表达系统给世界各地的实验室，它看起来很管用。而以这个系统为起点可以在体外构建RISC的各种活性，这样就可以来研究RISC的其他特性。
Q：你能用通俗的语言总结一下你的文章的意义吗？
A：RNA 干扰（RNAi）是一种分布广泛的生物调节机制，也是一个对基础和应用研究都很强大的工具，同时它还是一种有着巨大潜力的治疗手段。它已经在实验生物学领域中掀起了风暴。生物中从真菌和苍蝇到植物和人类，RNAi都在控制基因表达中扮演了多重的关键角色。RNAi机制中研究的最彻底的就是通过剪切损毁作为蛋白合成模板的“信使RNA”来抑制基因表达。
然而直到现在到底是哪个分子剪刀在RNA干扰的过程中剪切信使RNA仍然不清楚。不过我们的研究使得这个问题的解决成为可能。我们提供了Argonaute蛋白的结构，这个结构的一段区域是另外一个叫做RNA水解酶H（RNase H）——它可以切割RNA——的分离图形。这个发现提示Argonaute可能在这个被称为RISC的分子机器中担任十分重要的“切片机”的功能。这个发现还可以提示siRNA是怎么样导引RISC切割mRNA的。
通过和我的同事Greg Hannon合作，我们在哺乳动物细胞中测试了这个机制。结果显示在人源细胞的结构中，Argonaute 2是“切片机”。但该结果是通过直接从人源细胞中纯化的蛋白所获得。我们发现不同人源Argonautes所进行的实验有显著差异，这可能是由于蛋白有少许不纯导致我们看到的活性不同。
因此我们决定必须在完全缺少RNAi系统的细胞中生产Argonaute 2，大肠杆菌是首选。证明大肠杆菌产生的蛋白质有活性是非常重要的第一步，然后我们很快认识到这提供了一个很适合测试许多已知或者未知的RNAi剪切活性特性的paired-down系统。我们自己在这项研究中也测试了一些。
Q：你是怎么开始这项研究的？在这个过程中你遇到困难了吗？
A：作为一个正在被揭开其面纱的的重要生物途径，RNAi 是一个让人兴奋的新领域。而很多这方面的工作都在冷船港的实验室完成。这些研究在短时间内就对生物学产生了巨大冲击。而让我印象最深刻的是这些研究从遗传学、生物化学、分子生物学和生物信息学结合中获得的成果。因此我们认为对结构生物学来说这也是一个伟大的机会，因为结构生物学往往可以推动很多生物化学及其机制的研究。事实上我们在这篇文章中关于Argonaute结构的研究也确实推动了这方面的生物化学工作。
编辑：西门吹血
作者: Leemor Joshua-Tor
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