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Based on an endogenous regulatory antisense system in Dictyostelium (M. Hildebrandt & W. Nellen, Cell, 69, 197-204, 1992), we try to identify the cellular components involved in the mechanisms controlling gene regulation by experimentally introduced and natural antisense transcripts and to compare these mechanisms with RNA interference (RNAi).
We have identified and characterized a nuclease which specifically digests double stranded (ds)RNA and produces ~23mers, similar to the products of RNAi (J. Novotny, S. Diegel, H. Schirmacher, A. Möhrle, M. Hildebrandt & W. Nellen, Methods in Enzymology, 2001, Vol. 342, 193-212). Gene constructs expressing dsRNA can mediate RNAi in Dictyostelium, i.e. posttranscriptional gene silencing accompanied by the production of sequence specific ~23mers. Similar to Neurospora, C. elegans and others, an RNA dependent RNA polymerase (RdRp) is essential for efficient RNAi in Dictyostelium. We have identified three RdRp homologs in the Dictyostelium genome but only the disruption of
one of them (rrpA) abolishes RNA interference.
In Drosophila, the RNase III related gene "dicer" has been shown to generate ~23mers from dsRNA. We have identified two dicer homologs in Dictyostelium and are currently investigating their involvement in RNAi. Interestingly, no ~23mers are found when rrpA is knocked out, even though the dsRNase activity is still functional in vitro. Likewise, no ~23mers are detectable when the RNAi construct is introduced into cells which do not contain a target gene (e.g. b-galactosidase). We conclude that small amounts of ~23mers are produced from the original dsRNA (RNAi construct). These are unwound and serve as primers for RdRp on the target mRNA thus generating a secondary double strand. (H. Martens, J. Novotny, J. Oberstrass, T.L. Steck, P. Postlethwait & W. Nellen (2002): RNAi in Dictyostelium: the role of RdRPs and dsRNase. Mol. Biol. Cell, 13, 445-453).
![<a class=]() RNAi model with RDRP" hspace=20 src="http://www.uni-kassel.de/fb19/genetics/projects/RNAI_MODELL_MIT_RDRP.gif" width=500 vspace=20> | |
The figure depicts our current model on RNAi function which is based on our own data and on results
obtained by others in the model systems C. elegans and Drosophila.
The aim of our investigations is to dissect the molecular machinery which mediates gene silencing by antisense RNA and RNAi and to see how this is related to the regulation by endogenous antisense RNA. Antisense RNA and RNAi apparently function via completely base paired dsRNA. It has been suggested that antisense effects are either caused by minor amounts of contaminating dsRNA in injection experiments or by dsRNA formed by hybridization of antisense RNA and mRNA in vivo. To test this assumption, we evaluated the molecular requirements of antisense mediated gene silencing in comparison to RNAi. Surprisingly, we found that all three RdRPs, RrpA, RrpB and DosA were needed to achieve antisense effects. Coexpression of RNAi and antisense RNA had synergistic effects while coexpression of RNAi with sense RNA abolished gene silencing. To explain this, we extended our model:
 RNAi_Antisense.gif" width=640> | |
Here, antisense RNA is recognized by RrpB and/or DosA as "aberrant" and serves as a template for one or both of the RdRPs. We propose that RrpB and/or DosA are primer independent, in contrast to the primer dependent RrpA. "Aberrant RNA" is a term used since several years, especially in the plant field, to describe an RNA which causes gene silencing. This could be excess mRNA, antisense RNA or truncated transcripts. Possibly, "aberrant RNA" has unusual structural features which may be targeted by RdRP.
In the model, aberrant antisense RNA is transcribed by RrpB/DosA, the resulting dsRNA is cleaved by Dicer to ~23mers (siRNA), these molecules are unwound and can serve as primers for RrpA on sense as well as on antisense RNA. In contrast to the regular RNAi mechanism, which only uses the sense cascade, the siRNA pool is filled by an additional antisense cascade.
It should be noted that our model does not require RISC, a ssRNase complex described in Drosophila, that obtains sequence specificity by siRNAs. The data also suggest that antisense RNA does not directly interact with the message but rather serves as an autonomous substrate for the generation of ~23mers.
Prokaryotic antisense systems depend on specific structural features. Hybridization of distinct hairpin loops (kissing complexes) initiate sense – antisense interaction and finally result in complex structures consisting (in the case of the plasmid copy number control system copA/copT) of three inter- and two intramolecular helices (E.G.H. Wagner, S. Altuvia & W. Nellen 1999, Encyclopedia of Life Sciences, Macmillan Reference Ltd, http://www.els.net ). We have introduced the E. coli copA/copT antisense system into Dictyostelium and have preliminary evidence that not the completely double stranded hybrid but rather the formation of the five helix structure mediates gene silencing. This appears to be different from the RNAi mechanism described above. With the information available on antisense and RNAi mechanisms in Dictyostelium we will now be able to investigate if there are really two mechanistically distinct pathways for RNA mediated gene silencing.
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