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

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, ARF3͞ETT, 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.auxin response factor ͉ microRNA E ndogenous noncoding small RNAs 20-25 nt in length are important regulators of gene expression in both plants and animals (1-4). Recently, two major classes of small RNAs, microRNAs (miRNAs) and short-interfering RNAs (siRNAs), have been extensively studied. These two classes of small RNAs are generated from different types of precursor molecules, are processed through distinct biochemical pathways, and function in different biological contexts. miRNAs are processed from precursor molecules that are capable of forming double-stranded RNA (dsRNA) by intramolecular pairing (5) and act as regulatory factors during growth and development (1, 6, 7). In contrast, siRNAs are processed from either long bimolecular RNA duplexes produced by RNAdependent RNA polymerases (RDRs) or extended hairpins and function in several epigenetic and posttranscriptional silencing systems (8, 9). Two siRNA-generating pathways have been described: (i) an endogenous siRNA-generating pathway that requires RDR2 and triggers changes in the chromatin state of elements from which they derive (10) and (ii) an exogenous siRNA-generating pathway that requires RDR6 and targets viral RNAs (3). Despite the difference in the origin and generation of their precursors, both siRNAs and miRNAs require DICER proteins for processing, and both are assembled into the RNA-induced silencing complex (RISC) to target their complementary RNAs (3).Extensive efforts are being made to identify regulatory noncoding small RNA sequences, primarily focusing on miRNAs. Many miRNAs have been detected by using an established cloning strategy (11-14), although the low abundance and tissue-specific expression patterns of some make experimental identification difficult. Thus, different computational approaches have been developed to predict miRNA sequences based on t...

The Function of RNAi in Plant Development

“…Similar results for these genes were obtained by Vazquez et al (2004). Subsequent studies by us (Yoshikawa et al 2005) and other workers Williams et al 2005) revealed that most of the other up-regulated genes identified in this microarray analysis are also targets of siRNAs from unrelated transcripts and showed that the elevated expression of these genes in zip, rdr6, and sgs3 is correlated with the absence of these "trans-acting" siRNAs.…”

“…TAS1 and TAS2 do not appear to be conserved outside Arabidopsis thaliana. TAS3 regulates three members of the ARF family of auxin-related transcription factors (ARF2, ETT/ARF3, and ARF4) and is conserved throughout higher plants Williams et al 2005). …”

“…In contrast, siRNAs are the by-product of transcriptional or posttranscriptional silencing pathways, and in plants were thought to function primarily to suppress the expression of potentially deleterious RNAs, such as RNA derived from viruses and transposons. However, recent studies have revealed an unexpected role for RNAi in developmental gene regulation in plants and have led to the discovery of a new class of endogenous siRNAs, termed trans-acting siRNAs (ta-siRNAs) (Peragine et al 2004;Vazquez et al 2004;Allen et al 2005;Williams et al 2005;Yoshikawa et al 2005). ta-siRNAs are interesting not only because of their regulatory function, but also because their biogenesis is dependent on miRNA-directed cleavage of a precursor transcript Yoshikawa et al 2005).…”

Fine tuning of auxin signaling by miRNAs

“…The target is specified by the perfect or near-perfect sequence complementarity between the miRNA and its respective binding site on the targeted mRNA (Aukerman and Sakai, 2003;Wang et al, 2005a;Williams et al, 2005b). Experimental evidence indicates that there is usually a single miRNA binding site anywhere along the target mRNA.…”

“…Leaf development is regulated by modulating the expression of class-III homeodomain leucine zipper (HD-ZIP) transcription factor genes, which control leaf patterning along adaxial/abaxial axis (Juarez et al, 2004). PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV) are three closely related Arabidopsis HD-ZIP transcription factors that are the targets of miR165 and miR166 (Emery et al, 2003;Bao et al, 2004;Bowman, 2004;Juarez et al, 2004;Mallory et al, 2004b;Zhong and Ye, 2004;Kim et al, 2005;Williams et al, 2005b;Ko et al, 2006). Altered expression of miR165 and miR166 resulted in leaf developmental abnormalities in Arabidopsis and corn (Juarez et al, 2004).…”