圣路易斯华盛顿大学的生物学家们已经在理解植物细胞里用短片断RNA沉默不必要或者额外基因的途径方面取得了重大突破。基本上，正如古语所说，眼见为实，他们已经能够在细胞内看见该途径发生的位置和发生的方式。
华盛顿大学生物学教授Craig Pikaard博士和他的合作者已经叙述了拟南芥中8种蛋白在带来DNA甲基化途径中所起的作用，这种DNA甲基化是一种外成的作用，包括DNA四种碱基之一的胞嘧啶的化学修饰。在DNA不恰当甲基化的情况下，从植物到人的高等生物将会遇到许多发育问题：从植物的矮化病到人类的肿瘤、老鼠的死亡等。DNA甲基化的一个作用就是关闭一些重复的基因，例如一些在不加抑制的情况下能转移或者延伸到基因组的其他地方去阻断基因的转座子。人们对DNA甲基化研究产生重大的兴趣是由于它能帮助我们理解一些基因是怎么样被选择性的沉默的和沉默的等位基因怎样在将来的某一天重新开启，这些都具有现实意义。例如，肿瘤抑制基因在正常情况下帮助细胞保持正常分裂，但是在癌细胞中经常被DNA甲基化和组蛋白的修饰所沉默掉，导致了肿瘤的生长。和一些血液紊乱疾病是由于一些基因表达的缺失导致的，这些基因在人体发育早期表达，而在成体中遭受沉默，因此这些疾病的症状可以通过重新开启成年人的这些基因的表达得到减轻。
“我们正在研究的途径是植物发生的一个有趣现象的一部分，这种途径已经在人类中有所报道，叫做RNA介导的 DNA甲基化，”Pikaard解释说，“该途径发生在细胞核中，包括了一种短片断RNA，叫做小分子干扰RNAs——siRNAs。”
这些siRNAs，仅仅具有24个核苷酸的长度，负责甲基化与它自身序列配对的DNA序列，这种过程是在其他“朋友”的帮助下进行的。这些“朋友”是一组已知的RNA介导的DNA甲基化过程中的八个蛋白。
Pikaard和他的合作者利用一种熟练的技术不仅描述了该途径中的八种蛋白的位置，并且得到了导致甲基化的序列。这是一个曲折，但最终是一个循环的途径，Pikaard和他的合作者们是首次文字描述这些途径的研究人员并且提供了一种对导致甲基化的步骤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."
