Keywords: s. siRNA/RNAi; v. RNA transport and localization
In the last two decades many studies have utilized the powerful method of knocking down gene expression via RNAi. However, the level of knockdown varied between genes leading to a range of mRNA levels from full knockdown to very minimal decrease in the target mRNA. We found that two post-transcriptionally regulated mRNAs are protected in P-bodies, giving us a possible hint as to why certain mRNAs are not susceptible to RNAi. This hypothesis stemmed from our initial finding that oskar mRNA was only degraded after it localized to the posterior pole. oskar mRNA is transcribed and transported to the oocyte throughout oogenesis and it is localized to P-bodies. Since ribosome entry into P-bodies is inhibited, it is reasonable for us to hypothesize that at the posterior pole, where oskar mRNA is translated, the transcript has to be released from P-bodies, thus allowing access to the transcript for the RNAi machinery. Our finding that mRNAs localized in P-bodies are not degraded by RNAi came as a surprise to us. Numerous proteins that are components of the RNAi machinery localize into P-bodies, and we expected a strong knockdown when we initiated oskar RNAi. During early investigations into P-bodies, it was hypothesized that P-bodies are sites of mRNA decay due to the presence of numerous decay enzymes such as Dcp 1, Dcp 2 and Xrn 1. Recently, experiments carried out in live cells did not find mRNA degradation in P-bodies. Furthermore, it would be also of interest to assess if other translationally repressed mRNAs (i.e. nanos) are also protected from RNAi in P-bodies. Our results indicate that P-body protection is maintained in the nurse cells, but investigation of mRNA levels in the nurse cells could reveal further insights into this intriguing mechanism.