Mechanisms

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 RrPC (DosA) are required 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:

Here, antisense RNA is recognized by RrpB and/or RrpC (DosA) as "aberrant" and serves as a template for one or both of the RdRPs. We propose that RrpB and/or RrpC 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 RdRPs.
In the model, aberrant antisense RNA is transcribed by RrpB/C, the resulting dsRNA is cleaved by Dicer to ~21mers (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 ~21mers.

The detection of a new RNAi component, the RNA helicase HelF supported results decribed above. HelF is an endogenous inhibitor of RNAi and a gene knock-out significantly increased the efficiency of RNA interference. Surprisingly, PTGS by antisense RNA was not affected.
We found that the level of hairpin transcription (i.e. dsRNA) had a significant influence on RNAi mediated silencing efficiency: low amounts of transcribed dsRNA did not result in any silencing, intermediate amounts gave partial silencing and with high amounts, complete silencing was achieved. When HelF was knocked out, even low transcription levels of dsRNA were sufficient to obtain complete silencing. Unexpectedly, siRNAs were detected at similar quantities, independent of the levels of hairpin transcription, when gene silencing occurred. We assume that the siRNAs detected in Northern blots are rather secondary products of primed RdRPs on the target mRNA than primary products from the introduced hairpin construct.

This may be explained by a model where HelF serves as a surveillance system and mediates degradation of fortuitous dsRNA below a certain threshold level in the cell. Above the threshold, dsRNA is diced but the majority is degraded by the siRNA specific nuclease EriA. Only those molecules survive that are captured by RdRPs and guided to target mRNAs. RdRPs may be directly connected to Dicer and the polymerization product may be immediately processed to 21mers. These would then represent the siRNAs detected in Norther blots.

The model depicted below implies that primary and secondary siRNAs differ in structural or biochemical features. It furthermore suggests that primary siRNAs may be detected in a double knock out of HelF and EriA. Consitant with this model, EriA is upregulated in HelF KO strains and could thus degrade the larger amounts of hairpin RNA that are not abolished by HelF mediated degradation.

We are currently generating double KOs of HelF and EriA to support this assumption. In addition we try to isolate RNPs complexed with HelF, EriA and RdRPs to elucidate their interaction and to identify RNAs associated with these complexes.

Collaborations:
Michael Wassenegger Aiplanta, Neustadt, (http://www.agroscience.de/de/AlPlanta)
Fredrik Söderbom, SLU, Uppsala
Fritz HerbergKassel University, Kassel (http://www.biologie.uni-kassel.de/biochemistry)


Publications:

J. Novotny, S. Diegel, H. Schirmacher, A. Möhrle, M. Hildebrandt & W. Nellen (2001): dsRNAse in Dictyostelium, Methods in Enzymology, 2001, Vol. 342, 193-212

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

B. Popova, M. Kuhlmann, A. Hinas, F. Söderbom & W. Nellen (2006): HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing. Nucleic Acids Res., 34, 773-784, doi:10.1093/nar/gkj46

M. Kuhlmann, B. Popova & W. Nellen (2006): RNA interference and antisense mediated gene silencing in Dictyostelium. In: Dictyostelium discoideum. Methods in Molecular Biology. Edts.: L. Eichinger and F. Rivera. Humana Press.

M. Kuhlmann, B. E. Borisova, M. Kaller, P. Larsson, D. Stach, J. Na,  L. Eichinger, F. Lyko, V. Ambros, F. Söderbom, C. Hammann and W. Nellen (2005): Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi. Nucleic Acids Res. 33, 6405–6417, doi:10.1093/nar/gki952

M. Kuhlmann, B. Popova & W. Nellen (2005): RNA interference and antisense mediated gene silencing. In: Small RNAs: Analysis and Regulatory Functions . Series “Nucleic Acids and Molecular Biology”, Vol. 17, Edts.: W. Nellen and C. Hammann, Springer, I SBN: 3-540-28129-0