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

doi:10.1073/pnas.0504029102

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

United States Department of Agriculture–Agricultural Research Service, Plant Gene Expression Center, 800 Buchanan Street, Albany, CA 94710; and Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720

Communicated by Patricia C. Zambryski, University of California, Berkeley, CA, May 13, 2005 (received for review April 1, 2005)

Abstract

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.

Footnotes

↵ † To whom correspondence should be addressed. E-mail: fletcher{at}nature.berkeley.edu.
↵ * L.W., C.C.C., and K.S.O. contributed equally to this work.
Author contributions: L.W., C.C.C., K.S.O., and J.C.F. designed research; L.W., C.C.C., and K.S.O. performed research; L.W., C.C.C., K.S.O., and J.C.F. analyzed data; L.W., C.C.C., and K.S.O. wrote the paper; and J.C.F. revised and edited the manuscript.
Abbreviations: ARF, auxin response factor; miRNA, microRNA; siRNA, short-interfering RNA; dsRNA, double-stranded RNA; RDR, RNA-dependent RNA polymerase; RISC, RNA-induced silencing complex; tasiRNA, trans-acting siRNA; DCL1, DICER-LIKE1; RACE, rapid amplification of cDNA ends.