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First published online June 13, 2002; 10.1105/tpc.003210 American Society of Plant Biologists Endogenous and Silencing-Associated Small RNAs in PlantsCenter for Gene Research and Biotechnology, and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 1 To whom correspondence should be addressed. E-mail carrington{at}orst.edu; fax 541-737-3045
A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 sequences identified, the vast majority (90%) arose from intergenic regions, although small RNAs corresponding to predicted protein-coding genes, transposon-like sequences, and a structural RNA gene also were identified. Evidence consistent with the derivation of small RNAs of both polarities, and from highly base-paired precursors, was obtained through the identification and analysis of clusters of small RNA loci. The accumulation of specific small RNAs was regulated developmentally. We propose that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.
Eukaryotes have several types of tiny regulatory RNAs (Ruvkun, 2001  ; Waterhouse et al., 2001; Grosshans and Slack, 2002). Small temporal RNA (stRNA) and small interfering RNA (siRNA) are involved in the post-transcriptional control of gene expression. The stRNAs, such as those encoded by the Caenorhabditis elegans lin-4 and let-7 genes, bind to target mRNAs to repress translation (Lee et al., 1993; Moss et al., 1997; Olsen and Ambros, 1999; Reinhart et al., 2000; Slack et al., 2000; Grosshans and Slack, 2002). The siRNAs are associated with RNA silencing (or RNA interference [RNAi]), serving as guides for sequence-specific nucleolytic activity of the silencing-associated RISC complex (Hamilton and Baulcombe, 1999; Hammond et al., 2000, 2001; Zamore et al., 2000). The stRNA and siRNA are 21 to 25 nucleotides in length and arise from precursor molecules with extensive base-paired structure (Carthew, 2001; Ruvkun, 2001).
The functions of RNA silencing and stRNA-mediated repression of translation are partly understood. RNA silencing in plants is an antiviral response triggered by most or all viruses (Carrington et al., 2001
In C. elegans, RNAi serves to suppress the activity of several classes of transposons (Ketting et al., 1999
Both stRNAs and siRNAs arise by cleavage of precursor molecules containing exclusively or extensive duplex secondary structure by an RNaseIII-like enzyme (Bernstein et al., 2001
The relevance of small RNAs may be much broader than was suspected previously. Recent analyses of C. elegans, Drosophila, and human revealed dozens of small RNAs termed micro-RNAs (miRNAs) (Lagos-Quintana et al., 2001 In this study, a large set of small RNAs was identified in Arabidopsis. The majority of these RNAs arose from intergenic sequences, although several were identified from coding sequences and introns of conventional genes and transposable elements. The Arabidopsis small RNA populations may be more diverse structurally and functionally than the animal small RNA populations.
Amplification and Analysis of siRNAs from Plants Using a Model RNA-Silencing System The primary goal of this study was to explore naturally occurring, endogenous small RNAs of plants. However, siRNAs induced in a transient RNA-silencing system were analyzed initially for two purposes. First, siRNAs formed using defined RNA-silencing inducers provided a relatively simple model to adapt and refine methods for analyzing small RNAs in plants. Second, determination of the chemical nature of siRNAs in plants was necessary for comparison with endogenous small RNAs. Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens cultures containing expression cassettes for transcripts encoding green fluorescent protein (35S:GFP) or transcripts corresponding to intron-spliced, inverted-repeat forms of the GFP sequence (35S:dsGFP) (Figure 1A) .
The 35S:GFP and 35S:dsGFP constructs are weak and strong inducers, respectively, of RNA silencing in the infiltration zone (Johansen and Carrington, 2001 ). The GFP-related siRNAs from the infiltration zone were analyzed by blot assay using a radiolabeled GFP-specific probe. The siRNAs produced using either inducer migrated as a heterogeneous population with two predominant bands of  21 to 22 and 24 to 25 nucleotides in length (Figure 1B). The quantity of siRNA induced by the 35S:dsGFP construct was 20-fold greater than that induced by the 35S:GFP construct. Additionally, the ratio of the two major bands differed between siRNAs triggered by the two inducers, with the slower migrating band accumulating to relatively high levels using the 35S:dsGFP inducer (Figure 1B). The siRNAs were gel purified and ligated to RNA oligonucleotide adapters using T4 RNA ligase (Figure 2A) . Adapter 1 (A1) contained hydroxyl groups at both 5' and 3' ends and could ligate only to the 5' end of siRNAs. Adapter 2 (A2) contained 5' monophosphate and 3' inverted deoxythymidine groups and could ligate only to the 3' end of siRNA molecules. Intermediate and final ligation products using siRNAs were analyzed by RNA gel blot assay using a radiolabeled GFP-specific probe that detected both sense and antisense sequences (Figure 2B). siRNA self-ligation control reactions yielded products with electrophoretic mobilities consistent with the formation of siRNA dimers and larger concatemers (Figure 2B, lane 2). Ligation of siRNAs with the A1 adapter yielded major products with mobilities consistent with the joining of one siRNA and one adapter molecule (Figure 2B, lanes 3 and 5) and minor products with slower electrophoretic mobilities. The major A1-siRNA (using both inducers) ligation product was gel purified (Figure 2B, lane 6) and ligated to adapter A2.
The A1-siRNA-A2 ligation products were reverse transcribed, amplified by PCR, and cloned. The inserts in 72 recombinant plasmids containing GFP sequences (based on hybridization) were sequenced, revealing 59 unique siRNAs (Figure 2C and supplemental material). Nineteen sequences were from siRNA derived from the 35S:GFP inducer, whereas 40 sequences were from 35S:dsGFP-derived siRNA (Figure 2C). The siRNAs ranged from 20 to 25 nucleotides in length, with the most common lengths being 21 (44%) and 22 (25%) nucleotides (Figure 3A) . The distribution of siRNA sizes using the two inducers generally was similar, although the 24-nucleotide size class was recovered more often using the 35S:GFP inducer (26%) compared with the 35S:dsGFP inducer (13%). The relative proportion of 24-nucleotide siRNA in the sequenced populations was not reflected in the blot assay, in which siRNAs that comigrated with the 24-nucleotide standard were overrepresented using 35S:dsGFP (Figure 1B).
The unidirectional adapter ligation strategy allowed unambiguous determination of siRNA polarity. The ratio of sense-to-antisense siRNA was 8:11 using the 35S:GFP inducer and 20:20 using the 35S:dsGFP inducer (Figure 2C). Some clustering of siRNAs along the inducer sequences was detected. This was particularly evident for siRNAs of sense polarity induced by 35S:dsGFP (Figure 2C). Whether the apparent nonrandom distribution was caused by an amplification or sampling problem, or by a biologically relevant process such as preferential cleavage zones in siRNA precursors or siRNA stability, was not investigated. None of the siRNAs contained nontemplated 5' or 3' nucleotides. Although little or no bias was detected in the nucleotide composition of siRNAs, 5' guanosine and 3' thymidine were underrepresented among the total cloned sequences.
Small RNA Populations in Arabidopsis A total of 131 inserts were analyzed, yielding 125 unique Arabidopsis sequences (Table 1 and supplemental material). These ranged in length from 16 to 25 nucleotides, although the vast majority contained 21 to 24 nucleotides (Figure 3A). The overall size distribution of Arabidopsis small RNAs was similar to that of siRNA generated in the GFP- and dsGFP-silencing system, although the relative proportion of RNAs containing 21, 22, 23, and 24 nucleotides differed between the two sets (Figure 3A). Four of the small RNAs (15, 17, 33, and 125) contained single internal mismatches compared to the published Arabidopsis genome sequence (see supplemental data). Several lines of evidence argue against these sequences being formed as a result of the random breakdown of cellular RNAs.
The size distribution of the Arabidopsis small RNAs was coincident, over a narrow range, with that of the siRNAs (Figure 3A), even though the RNA substrates for adapter ligation were eluted from gels in the zone corresponding to 15 to 35 nucleotides. Also, the terminal structures of RNA fragments formed as a result of single-strand–specific ribonucleases typically contain 5' hydroxyl and 3' phosphate groups, which would prevent ligation to adapters. These data support the hypotheses that Arabidopsis contains large populations of small RNAs resembling siRNAs and that the small RNAs originate through processing by an RNaseIII-like mechanism. Although 125 unique sequences were identified, 539 genomic loci with sequence identity to small RNAs were mapped to IGRs, protein-coding genes, transposon-like sequences, and a structural RNA gene (Table 1, Figures 3B to 3D). Eighty small RNAs corresponded to unique loci, whereas 45 sequences corresponded to multiple loci (Table 1). Sequences from small RNAs were identified in each chromosome (Figure 3B), with the numbers of small RNA loci on chromosomes I, II, and IV being approximately proportional to chromosome size (Figure 3C). The numbers of loci on chromosomes III and V, on the other hand, were disproportionately high as a result of small RNAs 75 and 123, which were present in sequences that were repeated 64 and 137 times, respectively.
Small RNAs from Arabidopsis IGRs
Several small RNAs originated from each of five loci containing clusters of two to four small RNA sequences spaced irregularly within an IGR interval (Figure 4)
. In four of these clusters, the small RNA sequences overlapped partially. Interestingly, three of the clusters contained small RNA sequences of both sense and antisense polarities (Figure 4). The sequences surrounding and including each of the clustered small RNAs were predicted to fold into relatively stable base-paired structures containing up to 323 nucleotides (Figure 4). The predicted IGR structures generally were larger and more complex than those predicted to form in miRNA precursors in animals (Lagos-Quintana et al., 2001
The accumulation patterns of several IGR-derived small RNAs from the clustered loci were analyzed in replicated, normalized samples from Arabidopsis leaf, stem, and inflorescence tissue by blot hybridization using radiolabeled oligonucleotide probes. Discrete RNA bands that comigrated with either the 21- or 24-nucleotide standards were detected consistently using probes for small RNAs 19, 96, and 5 (Figure 5) . In each case, the electrophoretic mobility corresponded precisely with the size determined through sequence analysis. As shown in the blot for small RNA 5 (Figure 5), no slower migrating species corresponding to putative precursors were detected using any of the labeled probes. Each small RNA exhibited tissue specificity, with small RNAs 5 and 19 accumulating predominantly in leaves and small RNA 96 accumulating in inflorescence tissue. In addition, a small RNA band was detected in N. benthamiana leaf extracts that were probed for small RNA 5 (Figure 5).
Small RNAs from Arabidopsis Coding Regions and Introns Twenty-seven small RNAs (22%) corresponded to at least one genomic sequence of either sense or antisense polarity within a known or predicted protein-coding gene (Table 1, Figure 3D). Eight of these sequences corresponded to unique loci, and 19 were present at multiple sites (IGR or other protein-coding genes). Seventeen small RNAs mapped to sense or antisense sequences within predicted coding sequences, and 10 small RNAs mapped to sense or antisense intron sequences.
Nine small RNAs corresponded to genes resembling transposases or other proteins commonly found in transposable elements (Table 2). These included both long terminal repeat (LTR) and non-LTR retrotransposons (class I) and MULE and CACTA-like transposons (class II). The percentage (33%) of small RNAs mapping to transposon-like genes relative to the percentage corresponding to nontransposon protein-coding genes is substantially higher than that predicted based on the number of class I and II transposons in the Arabidopsis genome (Arabidopsis Genome Initiative, 2000
Small RNAs containing sense or antisense sequences of 16 nontransposon protein-coding genes were identified (Table 3). However, ESTs or cDNA clones have been identified for only four of these predicted genes. For two genes, which were predicted to encode a Ser carboxypeptidase–like protein and a protein of unknown function, multiple small RNAs were identified.
Two of the protein-coding genes were predicted to yield mRNA or pre-mRNA with unusual secondary structures. The small RNA 2 sequence was identified at two positions, one corresponding to a sequence annotated as IGR and the other corresponding to an antisense segment in the 3' proximal coding sequence of a predicted gene of unknown function (Figure 6 , Table 3). These two small RNA loci were adjacent to one another on chromosome V. However, the sequences surrounding the two small RNA sites contained extensive complementarity, which would yield an exceptionally stable stem structure in an mRNA precursor (Figure 6A). Small RNAs corresponding to sense and antisense small RNA 2 were detected by blot assay, and both accumulated predominantly in inflorescence tissue (Figure 6B). This region of chromosome V also contained a cluster of two other overlapping small RNA sequences (30 and 71).
At a Ser carboxypeptidase–like gene, small RNA sequences 90 and 101 were identified at two sites, separated by 41 nucleotides, within the predicted coding sequence. Complementary sequences to both small RNA 90 and 101 also were identified in a predicted intron for this gene. Secondary structure prediction indicated that a significant portion of the putative pre-mRNA would form an extensive base-paired stem composed of both coding and intron sequences (data not shown). These data indicate that at least some of the small RNAs from protein-coding genes are associated with sequences capable of forming extensively base-paired structures.
Several additional small RNAs mapped to both IGR and protein-coding genes. Small RNA 39 was identified at four positions in the Arabidopsis genome, including a chromosome III IGR sequence with the potential to form a stable stem structure (Figure 7A)
. This RNA also corresponded to an antisense segment from the central region of three closely related genes (Figure 7B), at least two of which are expressed. These genes belong to the Scarecrow-like (also known as GRAS) family of plant-specific transcription factors, >30 of which are predicted in Arabidopsis (Figure 7C). These factors are involved in a number of signaling and developmental processes, including the control of radial cell divisions in roots (SHR and SCR1), hormonal signaling by gibberellin (GAI1 and RGA1), and light signaling (PAT1) (DiLaurenzio et al., 1996
Small RNAs from Arabidopsis Structural RNA Two small RNAs (75 and 123) were clustered in or around the repeated 5S rRNA gene. The 5S rRNA gene is repeated within large head-to-tail arrays on three chromosomes (III, IV, and V), the typical repeat unit ( 500 nucleotides) being composed of transcribed sequence (120 nucleotides) and flanking spacer sequence (Cloix et al., 2002). The small RNA 123 sequence was identified in the sense orientation within the 5S transcribed region in 116 of the chromosome III repeats and 21 of the chromosome V repeats. This sequence overlapped box A of the internal promoter. The small RNA 75 sequence was in the opposite polarity relative to small RNA 123 and mapped to the spacer sequence on the 5' side of the transcribed sequence in 64 of the repeated units. All of the small RNA 75 sequences were in the centromere-proximal 5S rRNA array on chromosome III. This array was shown to be inactive in Arabidopsis (Cloix et al., 2002).
Endogenous Small RNAs in Arabidopsis A diverse population of small RNAs in Arabidopsis was identified in this study. Although these were isolated from plants that lacked any exogenous inducers of RNA silencing, the Arabidopsis small RNAs resembled siRNAs triggered by two types of RNA-silencing inducers in N. benthamiana. The endogenous small RNAs of Arabidopsis and siRNAs of N. benthamiana were clearly related based on similarity of size (predominantly 21 to 24 nucleotides in length) and inferred terminal structure, which strongly suggest that both types of RNAs are formed by an RNaseIII-like mechanism. Importantly, the Arabidopsis small RNAs also resemble miRNAs of animals, which in C. elegans and Drosophila are likely formed through the activity of Dicer (Hutvagner et al., 2001 ; Ketting et al., 2001; Knight and Bass, 2001; Lee and Ambros, 2001). Arabidopsis contains nine predicted genes with at least one RNaseIII-like domain similar to that in Dicer (Z. Xie and J.C. Carrington, unpublished data), although the roles of these genes in small RNA biosynthesis have yet to be assigned. Among the 125 endogenous small RNAs identified in this study, only two were identified in more than one clone, suggesting that the screen was far from saturating. Further adding to the complexity is the likelihood that the production of small RNAs is under developmental control. Small RNA 39, for example, accumulated to detectable levels only in inflorescence tissue. Like the mRNA transcriptome, the composition of small RNA populations is unique in different cell types and tissues.
Small RNAs from IGR Sequences
These distinct properties of Arabidopsis IGR small RNAs can be explained by a biosynthetic mechanism that includes transcription of IGR DNA to form a highly base-paired precursor, Dicer-like cleavage to yield limited small RNAs, and RNA-dependent RNA polymerase–mediated amplification of part of the precursor RNA. The initial and subsequent small RNAs would serve as primers in a PCR-like amplification mechanism, similar to that proposed for the amplification of target RNAs during RNAi in C. elegans and Drosophila (Lipardi et al., 2001
In fact, clustering of small RNAs in both orientations is precisely what happens during RNA silencing using either weak or strong inducers (Figure 2). Amplification of these IGR precursor transcripts to yield sequences in both orientations may be catalyzed by SDE1/SGS2, an RNA-dependent RNA polymerase–like protein that is required for RNA silencing triggered by transgenes and some viruses (Dalmay et al., 2000b What are the functions of IGR-derived small RNAs? First, at least some small RNAs may be similar to C. elegans stRNAs. These would anneal imperfectly to mRNA targets to modulate translation. The imperfect nature of the base pairing between small RNAs and target mRNAs, however, precludes the computer-based identification of interacting partners using available data. Second, the IGR small RNAs might interact with target mRNAs, as do stRNAs, but have effects on processes other than translation. These interactions could affect RNA splicing, mRNA localization or RNA turnover, depending on how the small RNA interacted with the factors that normally control these events.
Third, IGR-derived small RNAs with perfect or nearly perfect complementarity to an mRNA might trigger site-specific cleavage of the mRNA. This could happen if the small RNA incorporates into a functional RISC-like complex with sequence-specific nucleolytic function, as occurs during RNA silencing (Hammond et al., 2000
Fourth, some small RNAs may serve as guides for the modification of chromosomal DNA. RNA silencing is known to trigger cytosine methylation within homologous sequences in chromosomal DNA, and siRNAs have been proposed to function as guide sequences in this process (Jones et al., 1999
Small RNAs from Protein-Coding and Structural RNA Genes
Does the presence of this class of small RNA reflect the silencing of endogenous mRNA or structural RNA targets? In C. elegans, genetic analysis revealed that several factors required for RNAi also are required for transposon silencing (Ketting et al., 1999 In addition to small RNAs from transposons, the small RNAs from protein-coding gene sequences that are predicted to form extensive duplex structures may reflect RNA-silencing activity. Two such small RNAs, one corresponding to a Ser carboxypeptidase–like gene and one corresponding to a gene of unknown function, were detected by blot assay or identified in the cloned library (Table 3, Figure 6). As with the artificial 35S:dsGFP RNA-silencing inducer, extensive duplex structures in mRNA or pre-mRNA may be targeted efficiently for cleavage. These small RNAs then would function in the subsequent RNA silencing of homologous and closely related sequences.
Endogenous Small RNAs and Signaling
Cell-to-cell transport of small RNAs through plasmodesmata may enable signaling for post-transcriptional or epigenetic control in cells near sites of small RNA production. Long-distance transport of small RNAs through the phloem could contribute to certain types of regulation that occur systemically (Kuhn et al., 1997
Plants, Plasmids, Strains, and Agrobacterium tumefaciens Injection Nontransgenic Nicotiana benthamiana and Arabidopsis thaliana ecotype Columbia-0 were used. The 35S:green fluorescent protein (GFP) construct (pRTL2-smGFP) contained a functional copy of the soluble modified GFP coding sequence. The intron-spliced, inverted-repeat form of the GFP sequence (35S:dsGFP) construct (pRTL2-dsGFP) contained both sense and antisense soluble modified GFP sequences separated by a 120-nucleotide intron of the RTM1 gene (Chisholm et al., 2000 ), as described previously (Johansen and Carrington, 2001). These plasmids contained the 35S promoter and terminator sequences. Expression cassettes were inserted into vector pSLJ755I5, and the resulting constructs were introduced by triparental mating into Agrobacterium tumefaciens strain GV2260 (Johansen and Carrington, 2001).
Agrobacterium infiltration of leaves was performed as described (Llave et al., 2000
RNA Isolation and Blot Analysis
Blot hybridization analysis was performed as described (Llave et al., 2000 Densitometric analysis of at least four independent RNA gel blots exposed to x-ray film was used to assess the relative levels of small RNA accumulation. Synthetic oligoribonucleotides of 21 and 24 nucleotides, with 5' phosphate and 3' hydroxyl ends, were used as size standards during electrophoresis. Ethidium bromide staining and visualization of the 5S RNA/tRNA bands in small RNA gels were used to monitor the loading of RNA samples.
Amplification products from small RNA cloning experiments were resolved on 2% agarose gels in TBE buffer. Products from GFP-derived small RNAs were subjected to DNA gel blot analysis as described (Llave et al., 2000
Amplification and Cloning of Small Interfering RNAs from N. benthamiana The siRNAs were first ligated to the 5' RNA adapter 1. The ligation products were gel eluted and ligated to the 3' RNA adapter 2 as described above. Individual ligation reactions of siRNA to each adapter were performed as controls, and reaction products were monitored by electrophoresis on 15% polyacrylamide gels and RNA gel blots. The final ligation product then was used as a template in a reverse transcription reaction using the reverse primer (5'-ACTCTTGACGACTAC-3') and Superscript II reverse transcriptase (Bethesda Research Laboratories Life Technologies). This was followed by PCR using the reverse and forward (5'-CGTTTGCTGGCTTTG-3') primers and Taq DNA polymerase. Amplification products were purified by phenol-chloroform extraction and ethanol precipitation and then ligated into the T/A site of a pGEM-T Easy vector (Promega) using T4 DNA ligase (Promega). Electroporation-competent Escherichia coli DH10B cells (Bethesda Research Laboratories Life Technologies) were transformed with the siRNA library. Positive colonies were identified by PCR using the M13F (5'-CCCAGTCACGACGTTGTAAAACGACG-3') and M13R (5'-GCGGATAACAATTTCACACAGGAAACAGC-3') primers followed by DNA gel blot hybridization using a radiolabeled GFP probe. Plasmids were extracted from positive colonies, and the inserts were sequenced.
Amplification and Cloning of Small RNAs from Arabidopsis Small RNAs were named with numerical suffixes (e.g., small RNA 39). In cases in which a small RNA sequence corresponded to multiple genome loci, the loci number is indicated after the small RNA number (e.g., small RNA 39.3). Detailed information about each small RNA is provided in the supplemental material.
Computer Analysis of Small RNA Sequences from Arabidopsis
The potential secondary structure of RNA surrounding small RNA sequences was predicted using mfold version 3.1 (Mathews et al., 1999 Upon request, all novel material described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use in noncommercial research.
Accession Numbers
We thank Lisa Johansen, Stephen Chisholm, Zhixin Xie, and Robert Anderberg for valuable comments and advice during the course of this work. We also thank Ed Allen for constructing the Scarecrow gene family dendrogram. C.L. was the recipient of a postdoctoral fellowship from the Ministerio de Educacion y Cultura (Spain). This work was supported by Grants AI27832 and AI43288 from the National Institutes of Health and by Grant NRI 2002-35319-11560 from the U.S. Department of Agriculture.
Online version contains Web-only data.  Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003210. Received March 19, 2002; accepted April 15, 2002.
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Abstract
INTRODUCTION
-32P-dATP. DNA oligonucleotides complementary to small RNA sequences were end labeled with 
-32P-ATP using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Blots used for the analysis of small RNA sequences were prehybridized and hybridized using PerfectHyb Plus buffer (Sigma). Hybridization of blots using random-primed GFP probes was performed at 38°C, whereas hybridization of blots using oligonucleotide probes was performed at 20°C below the calculated dissociation temperature (Td) [Td (°C) = 4(G+C) + 2(A+T)]. Blots were washed at 50°C as described (Llave et al., 2000