RNA Interference in Caenorhabditis elegans : Mechanism and Application

Abstract

Viruses exploit their host by using many compounds of the host and cause damage by replication and spreading through the organism. Transposons are DNA elements that can move and multiply themselves within their host genome. As a result, they can cause damaging mutations. This thesis is about a defense mechanism against viruses and transposons, which is called RNA interference (RNAi). This mechanism is conserved in many organisms including plants, fungi, mouse, human and the fruit fly Drosophila. Much effort is used to unravel the mechanism underlying RNAi. Double stranded RNA (dsRNA) is the initiator of the process. First, the dsRNA is cleaved into small pieces (siRNAs). Next, these effecter molecules bind to RNAs. These RNAs are subsequently cleaved and degraded. Viruses and transposons can no longer replicate or spread due to the degradation of their RNAs. RNAi can also be applied to target specific RNAs. This is done to study gene functions. By analysis of the effects of RNAi mediated destruction of an mRNA, which results in loss of the protein, information on the function of a gene can be obtained. It is also possible to use RNAi to improve food products or in disease treatment. In order to use RNAi in the different applications it is necessary to know more about the mechanism. During my research I have identified components involved in RNAi. I have used the model organism Caenorhabditis elegans. This a small worm, which has been studied for many years by investigators of different specialities. Therefore, many details about this organism are known and numerous research methods are developed. Chapter 1 of this thesis gives an overview of what is currently known about the mechanism of RNAi. Chapter 2 is about an amplification step in the RNAi mechanism. RRF-1 is an enzyme implicated in this process. We propose that this enzyme produces new dsRNA using the RNA that has to be broken down as a template. This results in more effecter molecules that help to finish the RNAi process. Chapter 3 concerns RRF-3, a family member of RRF-1. RRF-3 seems to inhibit RNAi; removal of RRF-3 results in an increase in the efficiency of RNAi. This can be useful when RNAi is applied. We used the worms without RRF-3 to generate new data on the genes of C. elegans. This is described in chapter 4. Each mRNA was targeted using RNAi and the effects on the worms were determined. New information on approximately 400 genes was obtained. Another aspect of RNAi is the spreading of the process throughout the worm. The dsRNA that triggers RNAi (or a modified form) is able to spread; this results in mRNA breakdown in distant tissues. Chapter 5 discusses several components that seem to be involved in the spreading of RNAi in C. elegans.