Effective Small RNA Destruction by the Expression of a Short Tandem Target Mimic in Arabidopsis

Abstract

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.

Widespread Long Noncoding RNAs as Endogenous Target Mimics for MicroRNAs in Plants

“…Moreover, the expression level of miR160 was decreased markedly (Fig. 3E), in agreement with the previous report that some eTMs can induce miRNA degradation (Todesco et al, 2010;Ivashuta et al, 2011;Banks et al, 2012;Yan et al, 2012).…”

“…4, B and C), as well as accelerated flowering time (data not shown). These phenotypes resembled those of plants overexpressing artificial miR160 target mimics (Todesco et al, 2010;Yan et al, 2012). Correspondingly, the expression of known miR160 target genes was also significantly increased in the osa-eTM160-3 overexpression plants (Fig.…”

“…A previous report showed that when different sequences were used as backbones to design artificial target mimics for selected miRNAs, variations in inhibition efficiency existed (Ivashuta et al, 2011). Recently, another work identified that Short Tandem Target Mimic, which is composed of two target mimicry sites separated by a short linker, has a high efficiency to degrade miRNAs and exert target mimicry functions (Yan et al, 2012). These findings suggested that there may be some specific sequence motifs and/or spatial structures required for effective eTM functions.…”

“…Artificial TM designed with pairing patterns similar to that of IPS1 and miR399 have been proven to have the ability to cripple the functions of their corresponding miRNAs in transgenic plants (Todesco et al, 2010;Ivashuta et al, 2011;Yan et al, 2012). Recently, genome-wide screens of endogenous target mimics (eTMs) have been carried out in several plant species with fully sequenced genomes by bioinformatics methods and have identified some candidate eTMs (Ivashuta et al, 2011;Banks et al, 2012;Meng et al, 2012).…”

Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs

“…Regardless of their origin, the presence of low-abundance sequences raises the question of their biological relevance. miRNA depletion experiments have been carried out for conserved or highly conserved families, and have shown that such families play relevant roles during plant development 42,43 . However, depletion of low-abundance, non-conserved miRNA families has not yet been performed.…”