Use of intron-based RNA systems to study nuclear and cytoplasmic RNA interference processes in plants
Final Report Abstract
In this proposal, we investigated certain aspects of nuclear and cytoplasmic RNA interference (RNAi) in plants by using intron-based RNA systems as a tool to uncouple these two processes. Spliced introns are retained in the nucleus. Thus, by inserting RNAi triggers or targets in introns, we reasoned that they would necessarily be retained in the nucleus, allowing us to independently investigate nuclear from cytoplasmic RNAi. We had shown before that a RNAi trigger residing in an intron (intronic hairpin RNA, int-hpRNA) is inefficiently processed to short interfering RNAs (siRNAs), fails to trigger post-transcriptional gene silencing (PTGS) but nevertheless efficiently triggers RNA-directed DNA methylation (RdDM). Now, we found that an RNAi target residing in an intron (intronic sense RNA, int- RNA) is targeted for RdDM but not for PTGS, despite the presence of homologous siRNAs. These experiments revealed that (i) PTGS can be uncoupled from RdDM, (ii) RdDM can be established at the absence of PTGS, and (iii) siRNAs guide PTGS in the cytoplasm but not in the nucleus of plant cells. In another approach of this proposal, we tested the potential of inducing RdDM and transcriptional gene silencing (TGS) of endogenous promoters using our intron-based RNA system. For this reason, we compared the RdDM potential of a nucleusretained intronic hairpin RNAs (int-hpRNAs) and a polyadenylated cytoplasm-directed classical hpRNA. Our data showed that only the classical hpRNA was processed to detectable siRNAs but that both, the hpRNA and int-hpRNA triggered equally efficient RdDM of a tobacco endogenous promoter. However, no heterochromatinization and TGS was observed in either case. These data revealed that (i) classical hpRNAs are much more efficiently processed to siRNAs than int-hpRNAs, (ii) int-hpRNAs trigger RdDM but not PTGS, while classical hpRNAs trigger both RdDM and PTGS, and (iii) that endogenous promoters are receptive to RdDM but significantly resistant to TGS. Collectively, our studies using intron-based RNA systems may serve as a useful guide for additional future studies and/or applications where uncoupling of nuclear from cytoplasmic RNAi is desirable.
Publications
- Revisiting RNA-directed DNA methylation. RNA Biology 2013, 10, 453-455
Dalakouras, A., and Wassenegger, M.
- Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA. RNA Biology 2015, 12, 268-275
Dalakouras, A., Dadami, E., Bassler, A., Zwiebel, M., Krczal, G., and Wassenegger, M.
(See online at https://doi.org/10.1080/15476286.2015.1017216)
