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Proc Natl Acad Sci U S A. 2005 July 5; 102(27): 9703–9708. Published online 2005 June 24. doi: 10.1073/pnas.0504029102. | PMCID: PMC1172271 |
Copyright © 2005, The National Academy of Sciences Plant Biology A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes Leor Williams,* Cristel C. Carles,* Karen S. Osmont,* and Jennifer C. Fletcher† 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 Received April 1, 2005. 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. Keywords: auxin response factor, microRNA Endogenous 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 RNA-dependent 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 their secondary structure and evolutionary conservation ( 15– 18). In Arabidopsis, previously uncharacterized small RNAs were identified by two additional methods: microarray analysis ( 19) and the cDNA–amplified fragment length polymorphism technique ( 20). Both methods compared the RNA expression profile of wild-type plants with either various mutants in the small RNA pathways ( 20, 21) or with developmental mutants ( 19). To date, 114 loci have been identified from Arabidopsis that encode 45 distinct miRNAs ( www.sanger.ac.uk/software/rfam/mirna/index.shtml). However, the capacity to uncover previously uncharacterized miRNAs by using the above approaches seems to be largely exhausted, because many publications report the isolation of overlapping populations of miRNAs ( 3, 12, 15, 16); therefore, alternative approaches are needed. In a recent study by Yamada et al. ( 22) on the transcriptional activity of the Arabidopsis genome, 1,347 nonannotated ESTs and full-length cDNAs were found. Because these genes are located in intergenic regions and do not have an ORF, we thought it likely that some might correspond to potential regulatory RNAs. We thus considered this nonannotated EST database as a resource of as yet unreported small RNAs and developed a computational screen to exclusively identify miRNA/siRNA candidate sequences within it. The advantage of using this database is that it exclusively contains noncoding, nonannotated ESTs, allowing us to focus on only previously unreported candidate small RNA species. Our protocol allowed us to uncover in the database a previously characterized but unannotated miRNA as well as other putative previously uncharacterized small regulatory RNAs. Here, we report a previously uncharacterized small RNA species with sequence similarity to several AUXIN RESPONSE FACTOR ( ARF) genes. We show that the small RNA loci are present in trans to the target loci, and that both the small RNA and the putative target genes are conserved in maize and rice. Accumulation of this small RNA depends on both RDR6, which produces bimolecular RNA duplexes from single-stranded RNA molecules, and DICER-LIKE1 (DCL1), which processes double-stranded miRNA precursors into 21- and 22-nt mature single-stranded miRNAs. The characteristics of this small RNA are shared with a previously identified trans -acting siRNA (tasiRNA) ( 20, 21). We demonstrate that this tasiRNA targets mRNAs of three Arabidopsis ARF genes, ARF2, ARF3/ ETT, and ARF4, two of which were previously predicted to be regulated by small noncoding RNAs ( 21). Computational Analysis. The nonannotated, noncoding database used in this study contained a list of EST accession numbers ( 22). An algorithm based on java (Sun Microsystems, Mountain View, CA) (available upon request) was developed to automate the retrieval of the nonannotated, noncoding EST sequences from GenBank. Individual EST sequences were submitted to blastn software ( www.ncbi.nlm.nih.gov/blast) to identify short, nearly exact matches in the Arabidopsis genome. EST sequences aligning with protein-coding genes were analyzed with mfold 3.1 ( 23) and mirscan ( 24) to determine their secondary structure. Plant Materials and Growth Conditions. The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild-type reference. Homozygous rdr2–1, rdr6–15, and dcl1–9 mutant plants were in the Col-0 genetic background. All plants were grown under continuous, cool-white fluorescent light (120 μmol·m-2·s-1) at 22°C in a 1:1:1 mixture of perlite:vermiculite:topsoil and were watered with a 1:1,500 dilution of Miracle-Gro 20-20-20 fertilizer (Target, Berkeley, CA). RT-PCR. Total RNA isolated by using the RNeasy plant kit (Qiagen) was treated with RNase-free DNase (Roche) for 30 min at 37°C and then purified with phenol/chloroform. The first-strand cDNA synthesis was performed on 4 μg of total RNA by using Superscript III RNase H- reverse transcriptase (GIBCO/BRL) and specific primers to the sense or antisense tasiR-ARF precursor strands (och3S-p-tasiR-ARF 5′-GAGATTATTGGATCCGCTGTGC-3′ and och3AS-p-tasiR-ARF 5′-TGTGGAGATTAGCTCAGGAGGG-3′), according to the manufacturer's instructions. One of 20 μl of the reverse-transcription product was used for each PCR reaction. The annealing temperature was 54°C for all primer pairs, and 40 cycles of PCR were performed. RNA Gel Blot Analysis. Total RNA was isolated from frozen tissue and ground to powder in liquid nitrogen as described in ref. 25. Sixty micrograms of total RNA from the various genotypes was resolved in 15% polyacrylamide/7 M urea gels, electrotransferred to Zeta Probe membranes (Bio-Rad), and hybridized to a 21-nt antisense DNA oligonucleotide probe complementary to tasiR-ARF, as described in ref. 26. The tasiR-ARF probe was radioactively labeled with [α- 32P]dATP by using the StarFire oligonucleotide labeling system (Integrated DNA Technologies, Coralville, IA). The x-ray film (BIOMAX, Kodak) to which the membrane was exposed was developed after 12 days. Hybridization of the same filter by using a sense tasiR-ARF DNA oligonucleotide probe did not give any signal. The same RNA extracts also were probed with a [γ- 32P]ATP radioactively end-labeled DNA oligonucleotide probe ( 26) complementary to miR172b ( 27). In the case of miR172b, the x-ray film to which the membrane was exposed was developed after 3 days. 5′-Rapid Amplification of cDNA Ends (RACE). 5′-RACE was performed by using the Smart RACE kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions. Three rounds of PCR were performed by using nested primers (sequences available upon request). Products from the final round of nested PCR were cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced on a genetic analyzer sequencer (ABI Prism 3100, Applied Biosystems) to determine the cleavage sites for each ARF target gene. A Computational Screen to Identify Small RNAs. Plant miRNA loci encode initial transcripts called miRNA precursors, which are capped and polyadenylated and can appear as noncoding ESTs ( 28– 30). Thus, we reasoned that analysis of nonannotated, noncoding expressed sequences might reveal new miRNAs and retrieved a total of 1,347 Arabidopsis ESTs from a previously described nonannotated EST database ( 22). Our approach was to apply to each nonannotated EST a set of criteria that define previously described miRNAs. First, plant miRNAs have a high degree of complementarity with their targets, and the complementary site is typically located within the coding region of the target sequence ( 31). Therefore, each EST sequence was submitted to blastn searches against the Arabidopsis Genome Initiative database to identify short, nearly exact matches of 17–24 nt within the transcripts of coding genes. Positive hits were subjected to further analysis to determine whether they matched additional criteria. A second property of miRNAs is that they are located in intergenic regions and in trans to their target mRNAs ( 5). Consequently, the positive hits were confirmed to originate from noncoding, nonrepetitive genomic DNA loci. Third, miRNAs usually target members of a gene family ( 5); thus, we gave higher priority to sequences following this rule. Fourth, the majority of miRNAs are evolutionarily conserved ( 1, 32), so blastn searches were performed with the candidate sequences to identify potential matches in the rice genome. Finally, miRNAs are processed from precursors able to form hairpins ( 33). Therefore, the potential miRNA precursor sequences were submitted to the mfold and mirscan computer programs to predict their ability to form stem–loop structures ( 23, 24). Using this screening approach, we found an EST corresponding to a previously reported but not yet annotated miRNA (EST AU239920 corresponding to miR167d), thus validating our methodology. We identified several candidate sequences meeting most of our criteria: they are complementary to a protein-coding sequence, potentially target more than one member of a gene family, are transcribed from a noncoding genomic region, and are conserved among distantly related plant species. However, none of the candidates is predicted to form a hairpin structure. These findings suggest the existence of a different type of endogenous, small regulatory RNA. An Arabidopsis Noncoding EST Has a 22-nt Sequence Complementary to ARF Genes. Recently, two studies have provided evidence for the existence of a previously uncharacterized class of small RNAs in Arabidopsis ( 20, 21). Vazquez and coworkers described a set of endogenous siRNAs that are processed in an RDR6-dependent manner from a dsRNA transcribed from the noncoding At2g27400 gene. Like miRNAs, these previously uncharacterized siRNAs direct cleavage of endogenous mRNAs that have a region of complementarity to the siRNAs but little overall resemblance to the genes from which the siRNAs originated. However, unlike miRNAs, the production of these siRNAs requires the RDR6 and SUPPRESSOR OF GENE SILENCING3 (SGS3) proteins, which are components of the posttranscriptional gene silencing pathway. Moreover, the precursor molecule does not fold into a perfect hairpin. Because these siRNAs act in trans to cleave endogenous mRNA targets, these small RNA species were termed trans-acting siRNAs (tasiRNAs). Peragine et al. ( 21) independently demonstrated that RDR6 and SGS3 are required for the production of endogenous tasiRNAs, and that these small RNAs function in normal development. Using microarray analysis, this group found a small number of genes that are up-regulated in rdr6 and sgs3 mutants. Two of these genes, ARF3/ ETT and ARF4, encode members of a large family of auxin-regulated transcription factors, several of which are potential targets of miRNAs ( 26). Because ARF3/ ETT and ARF4 transcripts accumulate to higher than normal levels in rdr6 and sgs3 mutants, the data suggest that ARF3/ ETT and ARF4 are targets of negative regulation by small RNAs. However, Peragine et al. were unable to detect any small RNA sequence complementary to the coding region of these two ARF genes. In our computational screen, we identified two ESTs with two nearly identical, adjacent 21- and 22-nt sequences that are complementary to ARF3/ ETT and ARF4. This encouraged us to further analyze these ESTs as potential precursors of a tasiR-ARF. The two ESTs we identified in our screen correspond to the sense (EST AA651246) and antisense (EST AV534298) strands of a noncoding gene located on chromosome 3 (). Both ESTs were generated by using oligo(dT) primers, implying that both strands are polyadenylated. This nonannotated, expressed gene contains two exons and one intron (arrowhead). Apart from the fact that this sequence is not predicted to form a hairpin structure, it meets the other criteria we had designed for our computational screen. It has a 22-nt sequence that matches nearly perfectly (two mismatches) to the coding region of three members of the ARF gene family ( ARF2, ARF3/ ETT, and ARF4) (), and it is transcribed from a noncoding locus located in an intergenic region. Using this tasiR-ARF sequence to query the Arabidopsis genome, we found three additional ESTs from silique and inflorescence tissues. Two of these ESTs correspond to the sense strand and one to the antisense strand of the previously identified locus on chromosome 3. Two additional tasiR-ARF loci were found on chromosome 5, both carrying only one sequence complementary to the ARF genes (). We identified a corresponding EST for one locus only. We could not detect any evidence of expression for the second locus either in the databases or by RT-PCR (data not shown). tasiR-ARF and Its Target Genes Are Conserved in Rice and Maize. To determine whether the tasiR-ARF precursor is evolutionarily conserved, its sequence was queried against the rice and maize databases. In rice, four genomic clones with homology to the Arabidopsis tasiR-ARF precursor were found. Two of the clones contain two adjacent sequences complementary to the ARF genes, whereas the other two carry only one complementary sequence (). In maize, two mRNAs were found to contain putative tasiR-ARF complementary sequences; one has two adjacent sequences, and the other carries only one sequence (). We did not detect any similarity between the Arabidopsis, rice, and maize precursors outside of the region containing the putative small RNA. The ARF3/ ETT and ARF4 genes, which are up-regulated in rdr6 and sgs3 mutant plants ( 21), have two identical recognition sites for the putative tasiR-ARF (). Using blastn to search for additional target genes, we identified another member of the family, ARF2, that has a single 22-nt recognition sequence (). The alignment of the putative Arabidopsis tasiR-ARF sequence with the three ARF genes is shown in . In the rice genome, one ARF2-like gene and four ARF3/ ETT-like genes have one and two recognition sequences, respectively, each with two to three mismatches. In maize, two ARF-like genes each contain a single sequence that aligns with the tasiR-ARF, each with two to three mismatches (). The high degree of conservation of the 22-nt sequence in the tasiR-ARF precursor and in the potential ARF target genes between Arabidopsis, rice, and maize suggests that the proposed mechanism for ARF regulation by tasiR-ARF originated before the separation of the monocot and dicot lineages. These data suggest that tasiR-ARF is in this way similar to miRNAs, because most of the known Arabidopsis miRNA families are conserved between monocots and dicots ( 34). tasiR-ARF Accumulation Is RDR6- and DCL1-Dependent. We used RT-PCR to detect the expression of the predicted tasiR-ARF RNA precursor transcribed from the chromosome 3 locus. As shown in , both sense and antisense transcripts could be amplified from inflorescence and silique tissues, with a much higher level of expression observed in inflorescences. ARF3/ ETT and ARF4 are up-regulated in the rdr6 mutant ( 21), suggesting that the production of tasiR-ARF requires an RDR activity. We tested this hypothesis by using RNA filter hybridization and detected tasiR-ARF mature species in wild-type Col-0 and rdr2 mutant inflorescences but not in rdr6 or dcl1 mutant inflorescences (). A single band is detectable in the wild-type and rdr2 lanes, but we cannot say for certain whether both tasiR-ARF species accumulate or only one. Moreover, using RT-PCR analysis, we detected a higher level of the tasiR-ARF precursor transcripts in dcl1, compared with Col-0 (data not shown), indicating that the tasiR-ARF precursor dsRNA is not efficiently processed in the absence of DCL1. Taken together, the results indicate that the tasiR-ARF dsRNA precursor is generated through an RDR6-dependent siRNA pathway. However, unlike siRNAs, tasiR-ARF accumulation requires DCL1 (), an enzyme in the miRNA-generating pathway. This result demonstrates that the biogenesis of this small RNA involves components of both the miRNA- and siRNA-generating pathways. The weak signals obtained on the autoradiograph suggest that tasiR-ARF accumulates at a very low level (). To confirm this finding, we hybridized the same RNA samples with a miR172 end-labeled probe ( 27). We detected a strong signal for miR172 after 3 days of exposure to film. In contrast, we were able to detect tasiR-ARF only when using a StarFire-labeled probe, shown to be 10-fold more sensitive than an end-labeled probe ( 35), and exposing the membrane to film for 12 days. Furthermore, the fact that tasiR-ARF is not present in the Arabidopsis Small RNA Project database ( 36) is consistent with it being expressed at a very low level. However, the low expression of tasiR-ARF is not inconsistent with its potential function as a regulatory small RNA. The level of tasiR-ARF can be high in a very specific tissue or group of cells, and extracting RNA from the whole inflorescence might have diluted the tasiR-ARF RNA. An alternative scenario could be that the transcript level is elevated only under certain conditions, reflecting the hormone-responsive nature of the genes it targets. Our ability to identify the tasiR-ARF demonstrates the power of our database-scanning approach for identifying previously uncharacterized low-abundance small RNAs. tasiR-ARF Displays Properties Associated with Assembly into the RISC. Recent work has shown that the two strands of siRNA and miRNA duplexes differ in their ability to assemble into the RISC ( 37, 38). Typically, the 5′ end of the siRNA or miRNA, which displays U:A base pairing, is unstable because of the smaller number of hydrogen bonds and therefore preferentially enters into the RISC. This established functional asymmetry rule for siRNAs and miRNAs applies to tasiR-ARF: the 5′ end of tasiR-ARF contains four sequential U nucleotides, giving it a lower base-pairing stability than the G-rich 3′ end (). This observation implies that the tasiR-ARF is likely to assemble into the RISC, supporting the hypothesis that it functions through the siRNA pathway. The ARF Target Transcripts Are Cleaved in the 22-nt tasiR-ARF Recognition Site. Like miRNAs, the previously reported tasiRNAs from locus At2g27400 directed cleavage of their complementary mRNAs ( 39). To test whether tasiR-ARF directs cleavage of ARF2, ARF3/ ETT, and ARF4 mRNAs at the cognate recognition sites, 5′-RACE was performed (). Using primers specific for each ARF gene, the full-length transcripts for ARF2, ARF3/ ETT, and ARF4 were detected (, asterisks) as well as shorter species at the sizes expected for the cleavage products (, arrowheads). The three ARF genes always produced multiple bands in 5′-RACE reactions in repeated experiments with multiple templates and template concentrations. Several miRNA studies also obtained multiple bands in 5′-RACE reactions ( 21, 40). One interpretation for the multiple bands produced is that the mRNA of the ARF genes might tend to form secondary structures that sometimes cause reverse transcriptase to prematurely fall off of the mRNA, resulting in an incomplete cDNA. 5′-RACE products were cloned and sequenced to determine the precise sites of cleavage. All of the cloned sequences corresponded to ARF products. ARF2 has one tasiR-ARF recognition site, and ARF3/ ETT and ARF4 have two recognition sites (see ). Sequencing of the 5′-RACE products revealed multiple cleavage sites for ARF2, ARF3/ ETT, and ARF4 mRNAs within the recognition sequences (, arrowheads), as well as in close proximity to the recognition sequences. For example, of six ARF3/ ETT clones sequenced, two corresponded to transcripts cleaved after the 9th nucleotide, and one corresponded to transcripts cleaved after the 15th nucleotide. The other three clones corresponded to ARF3/ETT mRNAs that did not cleave directly in the recognition site but cleaved in close proximity to the site (data not shown). Cleavage at the canonical position relative to the 3′ end of the complementary target site was found for ARF2 only (, asterisk). Otherwise, the cleavage profiles of the ARF mRNAs differ from the canonical miRNA target cleavage profile of a single cleavage event after the tenth nucleotide ( 39). However, the existence of multiple cleavage sites as well as rare cases of cleavage outside of the recognition sequence have been previously reported for some miRNA targets ( 34). This result demonstrates that the ARF2, ARF3/ ETT, and ARF4 transcripts are subjected to negative regulation by cleavage and is consistent with potential regulation of these mRNAs by tasiR-ARF. The tasiR-ARF-Generating Pathway Involves Elements of both the miRNA and siRNA-Generating Pathways. The tasiR-ARF sequence that we have identified is an endogenous 21- and 22-nt sequence that belongs to a class of small RNAs called tasiRNAs. The tasiRNA biogenesis pathway corresponds to a fourth type of small RNA-generating system that involves factors required in two of the three systems previously reported in plants ( 10, 41). It involves DCL1, a miRNA-generating pathway component, and RDR6, a factor required by the exogenous siRNA-generating pathway (). Interestingly, although the tasiRNA pathway is endogenous, it does not seem to require RDR2 and DCL3, which are components of the endogenous siRNA pathway ( 20, 21). tasiRNA biogenesis differs from the above pathways in that both strands of the tasiRNA locus are transcribed. The existence of polyadenylated transcripts from both polarities does not rule out the possibility that tasiRNA precursors are both converted to dsRNA through RDR6 activity. The dsRNA can be generated from both strands and lead to the same small RNA product (, pathways  and  ). The genetic requirements for tasiRNA formation, which are intermediate between those of miRNAs and siRNAs, prompt questions about the evolutionary and functional specialization of these small RNAs. The diversification of small RNA-generating pathways has been proposed to contribute to the functional specialization of each small RNA species: miRNAs for development, endogenous siRNAs for chromatin modification, and virus-derived siRNAs for defense ( 10). The fact that we identified tasiRNA that potentially targets members of the hormone-responsive ARF gene family suggests a function for some tasiRNAs in development ( 42– 44), as proposed in ref. 21. Interestingly, in contrast to the At2g27400 tasiRNAs ( 20), tasiR-ARF is conserved in monocots, suggesting that this tasiRNA emerged quite anciently, before the divergence of the monocots and dicots that occurred 200 million years ago. Conservation among distantly related species is a common characteristic of miRNA families ( 11, 45, 46) but not of siRNAs, presumably because of the difference in their origin ( 10). Then, if tasiRNAs are as prevalent as other small regulatory RNAs, are most conserved between distantly related species, and do they all require the same set of biogenesis factors? The analysis of a larger group of tasiRNAs will help to address the question of whether the miRNA or the tasiRNA pathway is the more evolutionarily recent, or whether they evolved simultaneously from an ancestral pathway. Note Added in Proof. A trans-acting siRNA corresponding to tasi-ARF has been independently identified by Allen et al. ( 47) and named TAS3. We thank Kayoka Yamada and Athanasios Theologis for providing the nonannotated EST accession numbers, Stéphane Osmont for algorithm development, Marcey Sato for assistance with the database analysis, and Heather Fitzgerald and Jim Carrington for providing seed stocks. 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