Classification and functional annotation of endogenous siRNAs and other small RNAs

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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.