The non-coding Arabidopsis genome

我们过去的想法是这样的：我们的染色体是由DNA组成的，其中嵌入了我们的基因。当我们的基因被激活时，它们被转化为一种名为RNA的相关分子，它最终将我们基因的密码转化为蛋白质，执行我们细胞中的所有任务。我们染色体中的大部分DNA并不编码这样的基因，因此是垃圾。但我们现在的想法有点不同：尽管人类基因组在10年前就已经测序，但我们仍然在研究它的编码。令我们惊讶的是，更多的DNA被复制到RNA中，而不是蛋白质的编码。我们称之为非编码RNA，事实证明，许多非编码RNA在控制蛋白质编码基因的开启、关闭或修饰方面发挥着关键作用。我们现在意识到，为了了解基因和基因组是如何工作的，我们需要发现非编码RNA，并弄清楚它们的功能。我的实验室对植物如何控制花的发育很感兴趣。我们已经发现，非编码RNA控制着花的形成，这让我们对非编码RNA产生了兴趣。DNA测序最近通过所谓的下一代序列技术发生了革命性的变化。这种方法也可以用来对RNA的拷贝进行排序，使我们能够识别一个细胞中制造的所有RNA。RNA的拷贝是由一种叫做RT的东西制造的，所以我们几乎从来不直接看RNA。不幸的是，RT可能会犯错误，因此我们对RNA的解释可能是错误的。去年，一家名为Helicos Biosciences的公司开发了一种第三代测序技术，可以直接对RNA进行测序。我们已经与这家公司合作，对拟南芥(第一种完成整个基因组测序的植物)的RNA分子末端进行了测序。在这样做的同时，我们也发现了许多非编码的RNA，以及以前使用RT来查看RNA的实验存在的许多问题。在这项提议中，我们希望利用这项技术的突破来发现和注释拟南芥的非编码RNA。由于所有其他植物基因组的注释主要来自拟南芥，这对于理解其他植物基因组，包括我们赖以获得食物和燃料的作物，将是非常宝贵的。我们将寻找隐藏的RNA和长的非编码RNA。一些非编码RNA被破坏的速度几乎与它们产生的速度一样快，因此它们通常被有效地隐藏起来。但它们可以在缺乏外切体的植物中看到--外切体是一组破坏RNA的蛋白质。我们已经证明了在以前尝试查看这些RNA时存在许多错误，所以我们知道我们的方法是准确详细地显示这些RNA的一种很好的方法。我们已经进行的RNA测序发现了许多以前没有发现的RNA。问题是我们的测序只确定了这些RNA的末端，而我们不知道整个RNA分子的性质。最近对人类非编码RNA的一项研究发现，对RNA进行片断、测序和比较染色体上的特殊标记是有帮助的，这些标记可以发现基因的起始位置和基因的“主体”位置。我们将在这里对拟南芥采取同样的方法，但序列RNA直接复制，而不是RT复制。在这项提案中，我们还将尝试为我们发现的大量RNA确定功能。在人类中，长的非编码RNA充当了特殊蛋白质的支架，这些蛋白质可以关闭基因。我们将在拟南芥中找到与这些蛋白结合的RNA。我的实验室在这方面有特殊的专业知识，因为我们是第一批展示如何在拟南芥中做到这一点的实验室之一。最后，我们最近的突破确定了一组主要做得很好的非编码RNA：snoRNAs帮助修改其他RNAs。不幸的是，我们发现人们看待拟南芥非编码RNA的方式存在许多问题，这些问题可以用snoRNAs和RT错误来解释。为了防止未来发生这种情况，我们将整理这些RNA的注释。

What we used to think goes something like this: Our chromosomes are made of DNA and embedded within them are our genes. When our genes are switched on, they are converted into a related molecule called RNA, which eventually converts the code of our genes into proteins that carry out all the tasks in our cells. Much of the DNA in our chromosomes doesn't encode such genes and is therefore junk. But we think of things a little differently now: Although the human genome was sequenced 10 years ago we are still working out what it codes for. To our surprise much more of it is copied into RNA than that which codes for proteins. We call these non-coding RNAs and it turns out that many play critical roles in controlling how protein coding genes are switched on, off, or modified. We now realize that to understand how genes and genomes work, we need to discover non-coding RNAs and work out what they do. My lab is interested in how plants control flower development. We have discovered that non-coding RNAs control when flowers are made and this has got us interested in non-coding RNAs. DNA sequencing has been revolutionized recently by so-called next generation sequence technology. This approach can be used to sequence copies of RNA too, allowing us to identify all the RNAs made in a cell. Copies of RNA are made by something called RT, so we almost never look at RNA directly. Unfortunately RT can make mistakes so our interpretations about RNA can be wrong. Last year a company called Helicos Biosciences developed a 3rd Generation sequencing technology that can sequence RNA directly. We have collaborated with this company to sequence the ends of RNA molecules in Arabidopsis (the first plant to have its entire genome sequenced). While doing this we also found lots of non-coding RNAs and lots of problems with previous experiments that used RT to look at RNA. In this proposal we want to build on our breakthroughs with this technology to discover and annotate the non-coding RNAs of Arabidopsis. As the annotation of all other plant genomes largely derives from Arabidopsis, this will be invaluable in understanding other plant genomes, including the crops that we depend on for food and fuel. We will look for hidden RNAs and long non-coding RNAs. Some non-coding RNAs are destroyed almost as quickly as they are made, so they are normally effectively hidden. But they can be seen in plants that lack the exosome - a group of proteins that destroy RNAs. We have already shown there are lots of mistakes in a previous attempt to look at these RNAs, so we know that our approach is a great way to show these RNAs in accurate detail. The RNA sequencing we have already carried out identified lots of RNAs not previously found before. The trouble is our sequencing only identified the ends of these RNAs and we don't know the nature of the whole RNA molecule. A recent study of human non-coding RNAs found it helpful to fragment RNA, sequence, and compare special marks on chromosomes found where genes start and where the 'body' of a gene is. We will take the same approach here with Arabidopsis, but sequence RNA directly rather than RT made copies. In this proposal, we will also try to identify a function for a large number of the RNAs we find. In humans, long non-coding RNAs act as a scaffold for special proteins that switch genes off. We will find RNAs that bind to these proteins in Arabidopsis. My lab has special expertise here, as we were among the first to show how to do this in Arabidopsis. Finally, our recent breakthroughs identified a set of non-coding RNAs that mostly do a quite well worked out job: snoRNAs help modify other RNAs. Unfortunately, we found lots of problems in the way people have looked at Arabidopsis non-coding RNAs that can be explained by snoRNAs and RT mistakes. To prevent this happening in the future, we will sort out the annotation of these RNAs.