Effective Small RNA Destruction by the Expression of a Short Tandem Target Mimic in Arabidopsis[C][W]

This work presents a technology for effectively silencing endogenous small RNAs by expressing a small tandem target mimic (STTM) composed of two noncleavable small RNA binding sites linked by an empirically determined spacer. Expression of STTM in Arabidopsis thaliana leads to the specific degradation of endogenous small RNAs by small RNA degrading nuclease family enzymes. MicroRNAs (miRNAs) and other endogenous small RNAs act as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. The interrogation of small RNA functions requires an effective, widely applicable method to specifically block small RNA function. Here, we report the development of a highly effective technology that targets specific endogenous miRNAs or small interfering RNAs for destruction in Arabidopsis thaliana. We show that the expression of a short tandem target mimic (STTM), which is composed of two short sequences mimicking small RNA target sites, separated by a linker of an empirically determined optimal size, leads to the degradation of targeted small RNAs by small RNA degrading nucleases. The efficacy of the technology was demonstrated by the strong and specific developmental defects triggered by STTMs targeting three miRNAs and an endogenous siRNA. In summary, we developed an effective approach for the destruction of endogenous small RNAs, thereby providing a powerful tool for functional genomics of small RNA molecules in plants and potentially animals.