Classification and functional annotation of endogenous siRNAs and other small RNAs

该项目的目的是进行实验和编写计算机程序，以帮助研究哺乳动物细胞内产生的小干扰rna (sirna)（内源性sirna）的作用。sirna是由一种名为Dicer的蛋白质从细胞中相互结合的双链RNA分子的末端剪切而成的短RNA分子。释放的短双链片段被解开，其中一条链被装载到由一组其他蛋白质组成的结构中。siRNA链作为这些蛋白质的向导。siRNA在细胞内的其他rna中找到目标区域。如果这些区域沿着siRNA的整个长度与siRNA结合，结构中的一种蛋白质将切割目标，目标将被分解。如果siRNA的一端只有一小部分与靶标结合，靶标就会不稳定，蛋白质制造机器读取它的能力就会受到阻碍。通过这种方式，细胞中表达的sirna能够控制其他RNA的水平和由RNA信息制造的蛋白质的数量。在哺乳动物中，sirna被认为是罕见的，但技术的进步已经开始识别它们。然而，识别细胞中小rna的最常用方法只能找到它们，而不能预测它们的作用或它们是如何产生的。在同样的实验中也发现了许多其他的小rna，它们与sirna的长度相似，但可能具有不同的功能。为了弄清楚sirna在细胞中的确切作用，我们需要能够可靠地将它们与其他小rna区分开来。人们正在努力理解这些数据。RNA分子中的核苷酸序列可以用来追踪RNA分子，追溯到DNA基因组中它必须被读取的区域，一些工具可以根据这些信息识别出一些小RNA的类别。需要更多的工作来有效和自动地识别哪些小rna是内切sirna。这个提议的第一个目标是在实验室中生成数据，使我能够识别DNA基因组中表达dsRNA的区域，以及这些区域中哪些区域被Dicer切割以释放sirna。一旦我确定了这些区域，我将使用计算方法来寻找DNA中的局部特征和来自该区域的短rna的特征，以开发计算工具，可以从从细胞中采样的其他小rna中识别内切sirna。我还将研究产生其他类型小RNA的区域周围的特征，以寻找相似之处，并利用这些特征将短RNA分组成最有可能以相似方式表现的集合。最近在哺乳动物中也认识到，sirna能够通过直接靶向DNA来抑制来自基因组其他区域的RNA表达。这一现象发生的机制尚不完全清楚。同样，通过搜索可能被sirna靶向的区域周围的特征，我将通过实验来识别，我将努力开发一种方法，能够通过计算来预测DNA的区域，这些区域可能被内源性sirna靶向。预测的靶标将允许实验生物学家在他们自己的实验中跟踪有趣的候选靶标，并有助于未来对单个内源性sirna和这类小rna的作用的研究。越来越多的证据表明Dicer或其产生的内源性sirna对双链RNA的切片可能在多种发育过程和疾病中发挥作用。所产生的结果将有助于该领域的未来研究，并有助于更好地了解可能与人类健康和疾病相关的细胞过程。如果我们能更好地理解控制sirna靶向DNA区域的规则，sirna也有可能被用作药物。

The aim of this project is to perform experiments and write computer programs that will aid research into the role of small interfering RNAs (siRNAs) produced within mammalian cells (endogenous siRNAs). siRNAs are short RNA molecules snipped, by a protein named Dicer, from the end of double stranded RNA molecules which have bound to each other in the cell. The short double stranded segment that is released is unwound and one strand is loaded into a structure consisting of a set of other proteins. The siRNA strand acts as the guide for these proteins. The siRNA finds target regions in other RNAs in the cell. If these regions bind to the siRNA along its whole length, one of the proteins in the structure will cut the target and the target will break down. If only a small part at one end the siRNA binds to the target, the target will be destabilised and its ability to be read by the protein making machinery will be impeded. In this way siRNAs expressed in a cell are able to control the level of other RNAs and the amount of protein manufactured from the RNA messages.In mammals, siRNAs were thought to be rare but advances in technology have begun to identify them. However, the most frequent methods for identifying small RNAs in the cell can only find them and can't predict what they do or how they were produced. Lots of other small RNAs, that are a similar length to siRNAs but which may have different functions, have also been found in the same experiments. In order to work out exactly what siRNAs might be doing in the cells we need to be able to reliably distinguish them from other small RNAs.There are ongoing efforts to understand this data. The sequence of nucleotides in an RNA molecule can be used to trace the RNA molecule back to the region in the DNA genome it must have been read from and some tools exist that can identify some of the classes of small RNA based on this information. More work is required to efficiently and automatically identify which of the small RNAs are endo-siRNAs.The first goal of this proposal is to generate data in the laboratory that will allow me to identify regions of the DNA genome that express dsRNA and which of these regions are cut up by Dicer to release the siRNAs. Once I have identified these regions, I will use computational methods to look for local features in the DNA and features in the short RNAs from the area, to develop computational tools that can identify endo-siRNAs from amongst the other small RNAs sampled from cells. I will also investigate the features around regions that produce other types of small RNA to look for similarities and use these to group short RNAs into sets that are most likely to behave in a similar way.It has also recently been realised in mammals that siRNAs are able to suppress the expression of RNA from other regions of the genome by targeting the DNA directly. The mechanism by which this occurs is still not entirely understood. Again, by searching for features that surround regions potentially targeted by siRNAs, that I will identify experimentally, I will work to develop a method that will be able to computationally predict regions of the DNA that may be targeted by endogenous siRNAs in this way. Predicted targets would allow experimental biologists to follow up on interesting candidates in their own experiments and aid future research into the roles of individual endogenous siRNAs and this class of small RNAs in general.There is increasing evidence that the slicing of double stranded RNA by Dicer or the endogenous siRNAs produced may play a role in several developmental processes and diseases. The results generated will aid future research in this field and lead to a better understanding of the processes in the cell that may potentially be relevant for human health and disease. It is also possible that siRNAs could be used as medicines if we can better understand the rules that control the areas of DNA that they target.