圣路易斯華盛頓大學的生物學家們已經(jīng)在理解植物細胞里用短片斷RNA沉默不必要或者額外基因的途徑方面取得了重大突破?；旧?，正如古語所說，眼見為實,，他們已經(jīng)能夠在細胞內(nèi)看見該途徑發(fā)生的位置和發(fā)生的方式,。

        華盛頓大學生物學教授Craig  Pikaard博士和他的合作者已經(jīng)敘述了擬南芥中8種蛋白在帶來DNA甲基化途徑中所起的作用，這種DNA甲基化是一種外成的作用,，包括DNA四種堿基之一的胞嘧啶的化學修飾,。在DNA不恰當甲基化的情況下，從植物到人的高等生物將會遇到許多發(fā)育問題：從植物的矮化病到人類的腫瘤,、老鼠的死亡等,。DNA甲基化的一個作用就是關閉一些重復的基因，例如一些在不加抑制的情況下能轉(zhuǎn)移或者延伸到基因組的其他地方去阻斷基因的轉(zhuǎn)座子,。人們對DNA甲基化研究產(chǎn)生重大的興趣是由于它能幫助我們理解一些基因是怎么樣被選擇性的沉默的和沉默的等位基因怎樣在將來的某一天重新開啟,，這些都具有現(xiàn)實意義。例如,，腫瘤抑制基因在正常情況下幫助細胞保持正常分裂,，但是在癌細胞中經(jīng)常被DNA甲基化和組蛋白的修飾所沉默掉,，導致了腫瘤的生長。和一些血液紊亂疾病是由于一些基因表達的缺失導致的,，這些基因在人體發(fā)育早期表達，而在成體中遭受沉默,，因此這些疾病的癥狀可以通過重新開啟成年人的這些基因的表達得到減輕,。

        “我們正在研究的途徑是植物發(fā)生的一個有趣現(xiàn)象的一部分，這種途徑已經(jīng)在人類中有所報道,，叫做RNA介導的  DNA甲基化,，”Pikaard解釋說，“該途徑發(fā)生在細胞核中,，包括了一種短片斷RNA,，叫做小分子干擾RNAs——siRNAs。”

        這些siRNAs,，僅僅具有24個核苷酸的長度,，負責甲基化與它自身序列配對的DNA序列，這種過程是在其他“朋友”的幫助下進行的,。這些“朋友”是一組已知的RNA介導的DNA甲基化過程中的八個蛋白,。

        Pikaard和他的合作者利用一種熟練的技術(shù)不僅描述了該途徑中的八種蛋白的位置，并且得到了導致甲基化的序列,。這是一個曲折,，但最終是一個循環(huán)的途徑，Pikaard和他的合作者們是首次文字描述這些途徑的研究人員并且提供了一種對導致甲基化的步驟和基因沉默更清楚的理解,。

       英文原文：

Pathway toward gene silencing described in plants
 
Biologists at Washington University in St. Louis have made an important breakthrough in understanding a pathway plant cells take to silence unwanted or extra genes using short bits of RNA. Basically, they have made it possible to see where, and how, the events in the pathway unfold within the cell, and seeing is believing, as the old saying goes.

Craig Pikaard, Ph.D., Washington University professor of biology in Arts & Sciences and his collaborators have described the roles that eight proteins in Arabidopsis plants play in a pathway that brings about DNA methylation, an epigenetic function that involves a chemical modification of cytosine, one of the four chemical subunits of DNA. Without proper DNA methylation, higher organisms from plants to humans have a host of developmental problems, from dwarfing in plants to certain tumors in humans, and death in mice. One role of DNA methylation is to turn off repetitive genes, such as transposable elements that can move or spread throughout a genome and disrupt other gene functions if left unchecked. There is also interest in DNA methylation because understanding how some genes are selectively silenced and how silenced alleles can be turned on again may someday have practical benefits. For instance, tumor suppressor genes that normally help keep cells from dividing uncontrollably are often silenced by DNA methylation and histone (proteins that wrap DNA) modifications in cancer cells, contributing to tumor growth. And certain blood disorders resulting from defective genes expressed in adults might be alleviated if versions of those same genes that are only expressed very early in development, but are then silenced in adults, could only be turned on again.

"The pathway we are studying is part of an interesting phenomenon that occurs in plants, and reportedly in humans, too, called RNA-directed DNA methylation," Pikaard explained. "This pathway takes place in the nucleus, and it involves short RNAs, called small interfering RNAs -- siRNAs."

Those little tykes, just 24 nucleotides long, are somehow responsible for methylation of DNA sequences that match the sequence of the siRNAs, but not without a lot of help from their friends. The friends in this case are the team of eight known proteins of the RNA-directed DNA methylation pathway.

Using an impressive toolkit of sophisticated techniques, Pikaard and his collaborators not only have described the locations of the eight proteins in the pathway but also have provided the sequence of events that leads to methylation. It is a twisted, and ultimately circular path, but Pikaard and his colleagues are the first researchers to literally see the pathway and thereby provide a clearer understanding of the steps leading to methylation and gene silencing.

The results were published in the July 14, 2006 issue of Cell. The study was funded by the National Institutes of Health, Howard Hughes Medical Institute (HHMI) and Monsanto Company. Pikaard's collaborators include Olga Pontes, the first author of the study, other group members from his Washington University laboratory and the group of Steven E. Jacobsen, Ph.D., an HHMI investigator and professor of biology at the University of California, Los Angeles.

Using mutants, antibodies, and fluorescence microscopy techniques known as RNA fluorescence in situ hybridization (RNA-FISH) and DNA-FISH, Washington University postdoctoral researcher Olga Pontes, Ph.D., was able to unravel where the eight team players are located and in what order events in the RNA-directed DNA methylation pathway transpire. Using antibodies to detect the proteins, together with DNA-FISH to detect the DNA sites that give rise to the siRNAs, Pontes found that half of the team is located with the genes that match the siRNAs.

"The combination of DNA FISH and protein localization allowed us to say which proteins are sitting on the DNA that give rise to the siRNAs and also the loci that are modified by the siRNAs," Pikaard said.

Pontes found the other half of the team located within a special nuclear compartment known as the nucleolus, long known to be the production center for ribosomes. "She got a brilliant signal in the nucleolus, a brilliant dot in the same place for each of the proteins," said Pikaard. Using RNA-FISH, Pontes also found that the siRNAs were in that same dot within the nucleolus.

Pontes and Pikaard were able to deduce the order of events by studying mutations of all eight genes that give rise to the proteins, finding out what happens to the different proteins as the different genes are mutated, one by one. For instance, the researchers found the importance of RNA Polymerase IVa (Pol IVa) by looking at a Pol IVa mutant and noting that the rest of the proteins didn't localize properly. In the RNA-dependent RNA polymerase 2 (RDR2) mutant, Pol IVa is unaffected, but the function of all the other proteins downstream is lost, inferring that it came into the act second. The picture that emerged from this logical approach is that Pol IVa gets things started, churning out RNA that then goes to the nucleolus where it is acted on by RDR2, which turns the single-stranded RNA into double-stranded RNA. The Dicer-like 3 protein, DCL3 then chops the RNA into small interfering RNAs (siRNAs). Along comes ARGONAUTE4 (AGO4), which grabs hold of the siRNAs while also binding to NRPD1b, the largest subunit of an alternative form of RNA Polymerase IV, Pol IVb. The AGO4-siRNA-NRPD1b complex is then thought to leave the nucleolus, acquire the second-largest Pol IV subunit, NRPD2, which serves both Pol IVa and Pol IVb, and then seek out the DNA sequences that match the siRNAs. At these sites, the chromatin remodeler DRD1 presumably bulldozes histones and other proteins out of the way to make the DNA accessible for methylation by the de novo cytosine methyltransferase, DRM2.

A paradoxical aspect of the pathway is that siRNAs direct DNA methylation but DNA methylation is also required for the production siRNAs. "It's a circular pathway. You have to produce the siRNA in order to have them come back and methylate the loci, which somehow induces more siRNA production involving Pol IVa". Pikaard said.

A combination of genetic mutants, transgenes, antibodies, RNA-FISH and DNA-FISH were key to the study. "This toolkit is really powerful," Pikaard said.

"It enabled us to look at a complex pathway and figure out not only the order of events but also the spatial organization of the pathway in the nucleus. Our hope for the future is to develop tools that will enable us to watch the pathway function in live cells using fluorescent proteins and time-lapsed microscopy, to learn even more."