The Amplification Step of RNAi: Dissection of the Mechanism and Application in Mammalian Cells

Genomes are under constant attack of parasites like viruses and transposons, which is apparent from the fact that 50% of the human genome consists of remnants of these molecular parasites. Defence is vital. Gene-silencing appears to be the answer of genomes and these mechanisms are common for a large variety of organisms including plants, fungi and animals. Both during my PhD and the following 6 years as a post-doc, I choose to work on gene-silencing processes in both plants and animals and I followed and explored the new insights constantly raised within this rapidly developing field. An effective genome defence mechanism should meet two main requirements: it needs to be gene-specific as ,,self,, has to be discriminated from ,,non-self,, and it asks an amplification step to fight off multiplying parasites. RNA interference (RNAi) is such a defence mechanism and comprises sequence-specific degradation of RNAs. The sequence-specificity comes from the trigger which is double-stranded RNA (dsRNA). Cleavage by a dsRNase results in small interfering RNAs (siRNAs) that guide RNA turnover of homologous sequences. Very recently, we showed (Sijen et al, 2001, Cell 107, 465-476) that these siRNAs are amplified by the action of a cellular RNA-directed RNA polymerase (RdRP). Amplification is essential for establishing silencing as RdRP mutants do not show RNAi. I plan to study the siRNA amplification mechanism in more detail and exploit the position we have in the field. The process will be studied in Caenorhabditis elegans as this organism is an excellent model for both genetics and RNAi. In addition I plan to investigate whether we can enhance RNAi in mammalian cells by using components of the amplification machinery. RNAi in mammalian cells is still in its infancy but it holds great promise for applications in gene-specific therapeutics.