A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes

Two classes of small RNAs, microRNAs and short-interfering RNA (siRNAs), have been extensively studied in plants and animals. In Arabidopsis, the capacity to uncover previously uncharacterized small RNAs by means of conventional strategies seems to be reaching its limits. To discover new plant small RNAs, we developed a protocol to mine an Arabidopsis nonannotated, noncoding EST database. Using this approach, we identified an endogenous small RNA, trans-acting short-interfering RNA-auxin response factor (tasiR-ARF), that shares a 21- and 22-nt region of sequence similarity with members of the ARF gene family. tasiR-ARF has characteristics of both short-interfering RNA and microRNA, recently defined as tasiRNA. Accumulation of trans-acting siRNA depends on DICER-LIKE1 and RNA-DEPENDENT RNA POLYMERASE6 but not RNA-DEPENDENT RNA POLYMERASE2. We demonstrate that tasiR-ARF targets three ARF genes, ARF2, ARF3/ETT, and ARF4, and that both the tasiR-ARF precursor and its target genes are evolutionarily conserved. The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves our method to be a useful tool to uncover additional small regulatory RNAs.