Journal
PLANT JOURNAL
Volume 69, Issue 2, Pages 217-226Publisher
WILEY-BLACKWELL
DOI: 10.1111/j.1365-313X.2011.04783.x
Keywords
miRNA; trans-acting siRNAs; RNA-induced silencing complex; 3' UTR; synthetic trans-acting siRNAs; RNA interference
Categories
Funding
- National Science Foundation [MCB1110957]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1110957] Funding Source: National Science Foundation
Ask authors/readers for more resources
In plants, many mRNAs and non-coding RNAs are cleaved by RNA-induced silencing complexes. After cleavage, only a limited number of RNAs are processed into trans-acting siRNAs (tasiRNAs). One reason is that 22 nt small RNAs, but not the more common 21 nt small RNAs, can efficiently trigger tasiRNA formation. The characteristics of the target transcripts may also affect tasiRNA production. Here we report the effects of target site location and sequence complementarity on tasiRNA formation. A synthetic sequence that included a miR173 target site and two siRNAs targeting an endogenous mRNA encoding PHYTOENE DESATURASE3 was introduced into a protein-coding (GFP) gene in the coding region or 3' UTR. tasiRNAs were generated in the transgenic seedlings, and the PDS3 mRNA level was reduced, leading to a photobleaching phenotype. It was found that tasiRNAs were most efficiently produced when the miR173 target site was placed immediately after the stop codon. Introducing premature stop codons caused a dramatic reduction of tasiRNAs and over-accumulation of 3' cleavage products, suggesting positive effects of translation on processing the 3' cleavage products into tasiRNAs. By systematically mutating the miR173 target site, we found that perfect complementarity between the 3' end of miR173 and the 5' end of the target sequence was crucial. Mismatches at that position abolished tasiRNA formation, but mismatches at the 5' end of miR173 had less effect. These data suggest important roles for translation and specific sequence complementarity in tasiRNA formation, providing new insights into tasiRNA biogenesis as well as a strategy for improving the efficiency of RNA interference (RNAi) using tasiRNAs.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available