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First published online July 16, 2004; 10.1105/tpc.104.022830 © 2004 American Society of Plant Biologists Novel and Stress-Regulated MicroRNAs and Other Small RNAs from ArabidopsisInstitute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521 1 To whom correspondence should be addressed. E-mail jian-kang.zhu{at}ucr.edu; fax 909-827-7115.
MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. In Arabidopsis thaliana, 43 miRNAs comprising 15 families have been reported thus far. In an attempt to identify novel and abiotic stress regulated miRNAs and siRNAs, we constructed a library of small RNAs from Arabidopsis seedlings exposed to dehydration, salinity, or cold stress or to the plant stress hormone abscisic acid. Sequencing of the library and subsequent analysis revealed 26 new miRNAs from 34 loci, forming 15 new families. Two of the new miRNAs from three loci are members of previously reported miR171 and miR319 families. Some of the miRNAs are preferentially expressed in specific tissues, and several are either upregulated or downregulated by abiotic stresses. Ten of the miRNAs are highly conserved in other plant species. Fifty-one potential targets with diverse function were predicted for the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Arabidopsis. These findings suggest that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.
Whereas the roles of proteins as regulatory factors in development and adaptation to the environment are well known, the involvement of regulatory small RNA molecules is just emerging (Carrington and Ambros, 2003  ). MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are two classes of small noncoding RNAs with important roles in the regulation of gene expression in plants and animals. miRNAs are an abundant class of tiny RNAs that are often found to be conserved evolutionarily across species boundaries and are capable of regulating the expression of protein-coding genes in multicellular eukaryotes (Bartel, 2004). miRNAs are  20 to 24 nucleotides in length, single-stranded noncoding RNAs derived predominantly from intergenic regions. miRNA genes are transcribed as long pre-miRNA transcripts ranging from 70 to 300 nucleotides that form fold-back structures in which mature miRNAs reside in either the 5' or 3' arm and are processed by DICER (Ambros et al., 2003a; Bartel and Bartel, 2003; Bartel, 2004). miRNAs are known to silence genes posttranscriptionally by targeting cognate mRNAs for degradation (Llave et al., 2002a; Palatnik et al., 2003; Tang et al., 2003) or translation repression (Lee et al., 1993; Reinhart et al., 2000; Aukerman and Sakai, 2003; Chen, 2004).
The miRNA profile of Caenorhabditis elegans is probably the most complete, with the number of validated miRNA genes being
siRNAs are double-stranded 21- to 26-nucleotide small RNAs derived from the processing of long double-stranded RNAs (Waterhouse et al., 2001
A distinguishing feature of plants is that they are sessile and thus have to cope with, rather than move to avoid, adverse environments. Plants have evolved sophisticated mechanisms to adapt to environmental stresses (Zhu, 2002
Identification of 15 New Families of miRNAs from Stress-Treated Seedlings To identify miRNAs that might be regulated by abiotic stress, the expression of known Arabidopsis miRNAs under dehydration, salt, cold, and ABA treatments was tested by RNA gel blot analysis. However, we did not find substantial stress regulation on any of the known miRNAs (see Supplemental Figure C online; data not shown). Therefore, to identify novel miRNAs and miRNAs that might be regulated by abiotic stresses, we constructed a library of small RNA species from pooled seedlings exposed to cold, dehydration, salinity, and ABA. Small RNAs (15 to 26 bp) were recovered by size fractionation on denaturing polyacrylamide gels, eluted from the gel, ligated to 3' and 5' adapters, amplified, cloned, and sequenced. A BLAST search against the Arabidopsis database revealed that a major portion (90%) of the 2500 cloned sequences appears to be breakdown products from rRNAs, tRNAs, and small nuclear RNAs. The proportion (9%) of putative regulatory small RNAs is slightly lower than published results, presumably because the library was made from seedlings subjected to different abiotic stresses that may cause enhanced breakdown of noncoding RNAs (rRNAs, tRNAs, and nucleolar RNAs). Several clones matched to chloroplast or mitochondrial sequences and may represent degradation or possibly regulatory products of organellar RNAs. A few clones do not have perfect matches to the Arabidopsis genome sequence and appear to be derived from RNAs of fungal/bacterial/viral origin. Thirteen previously published families of miRNAs from Arabidopsis are represented in our sequences. These include miR156, miR157, miR158, miR159, miR161, miR163, miR166, miR168, miR169, miR171, miR172, miR173, and miR319/miRJAW (Llave et al., 2002b; Park et al., 2002; Reinhart et al., 2002; Palatnik et al., 2003). The frequency of any one miRNA appearing in our sequenced library varied from 1 to 30 times (Table 1; see Supplemental Table 1 online).
Importantly, we identified many new miRNAs and putative siRNAs. The ability of RNA corresponding to the genomic sequences surrounding the clones, to fold into hairpin miRNA precursors, was applied to distinguish miRNAs from other potential small regulatory RNAs. This criterion, together with phylogenetic conservation of mature miRNAs, is now generally accepted as evidence for the existence of a miRNA (Ambros et al., 2003a). We analyzed miRNA precursors of up to 300 nucleotides for hairpin structures using the mfold program (see Supplemental Figure A online) (Zuker, 2003 ). The analysis revealed that we have cloned 21 new miRNAs from Arabidopsis that correspond to 29 loci (Table 1). Furthermore, on analyzing the Arabidopsis genome, we were able to predict three homologs of the cloned miRNAs miR399b and miR399f based on sequence similarity, the capacity of their precursor sequences to adopt hairpin structures, and their conservation in rice (Oryza sativa) (Figure 1F). One homolog for each of the cloned miRNAs, miR389a.1 and miR407a, is also predicted in Arabidopsis (Figures 1C and 1G). Taken together, the total number of new miRNAs identified in this study is 26, which come from 34 loci. Twenty-four of the newly identified miRNAs define 15 new families (Table 1, Figure 1). Two of the new miRNAs, miR171b and miR319c, belong to the previously reported miR171 and miR319/miRJAW families, respectively.
Genomic Organization of the miRNA Genes Twenty of the miRNAs identified in this study, including the five predicted miRNAs, correspond to single copy loci in the Arabidopsis genome, whereas the other six miRNAs (miR399b and c, miR393a and b, miR398b and c, miR405a, b, c, and d, miR171b and c, miR389a.1, and miR389b.1) correspond to multiple loci (two to four) that are scattered in the genome (Table 1). Out of the 34 loci, 15, 9, and 5 come from chromosome 2, 1, and 5, respectively, and the remaining five loci are represented in chromosomes 3 and 4. Based on sequence homology and predicted fold-back structures, we were able to identify some miRNA homologs in rice, Lotus, Medicago, and Populus. Thirty-two miRNA loci matched with intergenic regions, whereas two loci correspond to either an intron or 3' untranslated region (UTR) of a gene. miR408 is located in the antisense polarity to the 3' UTR of At2g47020 (Table 1). miR402 originates from the intron of gene At1g77230, in the same orientation as the pre-mRNA. Small RNAs corresponding to the introns or exons of pre-mRNAs in the same polarity may be miRNAs or may simply be general degradation products. The miRNAs can be ascertained by the capacity of their precursors to form hairpin structures, together with expression studies. The miR402 precursor sequence has the ability to adopt a long and typical fold-back structure (see Supplemental Figure A online). In addition, this miRNA can be detected as a discrete band of 22 bp on RNA gel blots (Figure 5E). These results show that it is an miRNA and not a nonspecific degradation product from the intron.
miR399 and its homologs correspond to six genomic loci (a, b, c, d, e, and f; b and c are identical in sequence but come from two different loci [Table 1]) in Arabidopsis with identical sequences or only a slight change in sequence. A sequence identical to miR399f is also found in rice, but the rice precursor sequence does not appear to form a fold-back structure; therefore, it cannot be ascertained as miRNA. However, rice has nine other loci with sequences that differ from the Arabidopsis miR399f in only one or two nucleotides (Figure 1F). These six loci from Arabidopsis and nine loci from rice all have the ability to form fold-back structures. Arabidopsis miR399 loci, miR399f, miR399d, and miR399e are located within a 2.0-kb region (Figure 2), and miR399f and miR399d are oriented in opposite directions. This organization of the two miRNAs within 2.0 kb and in opposite orientations is also conserved in rice (Figure 2). The only difference is that an additional locus miR399e exists in Arabidopsis in the same orientation as miR399f, but this is absent in rice. The conserved arrangement of these miRNAs suggests that some important aspects of their regulation may also be evolutionarily conserved.
Although miR389 has three loci in Arabidopsis, only two loci (on chromosomes 1 and 2) are able to adopt fold-back structures (Figures 3A and 3B). The predicted fold-back structure of miR389b.1 from chromosome 2 may give rise to two miRNAs that belong to the same family, one each from the 5' and 3' arms (Figure 3B). However, only one of the two (from the 3' arm, miR389b.1) is represented in the library, and the other (on the 5' arm, miR389b.2) is predicted based on sequence identity. Because miR389b.1 and its predicted homolog on the 5' arm are not located directly opposite to each other (Figure 3B), this represents a novel type of miRNA gene organization.
Conserved miRNA Gene Families Based on sequence similarity, the miRNAs were grouped into families. Five of the miRNA families (miR399, miR393, miR398, miR171b, and miR408) are highly conserved in rice (Table 1, Figure 1). Some are also conserved in Lotus and/or Medicago or Populus (Table 1, Figure 1). The miR399 family is represented by six members in Arabidopsis and nine members in rice (Figure 1F) and is one of the largest miRNA families in plants. miR171b is represented by one and seven members in Arabidopsis and rice, respectively, with slight changes in nucleotide sequence (Figure 1A). The miR398 family has two members each in Arabidopsis and rice (Figure 1E). miR393 is represented by one member in Arabidopsis and two members in rice (Table 1, Figure1D). Two members of miR389a.1 and miR407 with slight change in sequence exist in Arabidopsis (Figures 1C and 1G). In some cases, the number of family members is greater in rice than in Arabidopsis. For example, miR156 (Reinhart et al., 2002 ) and miR171b and miR399 (this study) are represented by more family members in rice.
One miRNA (miR319c) was represented by five clones in our library, and the sequence is highly similar to the previously cloned miR319/miRJAW and miR159 (Reinhart et al., 2002
Predicted Targets of the miRNAs
Our analysis predicted 51 potential targets for 21 of the newly identified miRNAs (Table 2). The plant miRNA target sites are predominantly found within the protein-coding segment of the mRNAs (Rhoades et al., 2002 ), whereas animal miRNAs appear to primarily target the 3' UTRs (Enright et al., 2003; Lewis et al., 2003; Stark et al., 2003). Consistent with the earlier findings in Arabidopsis (Rhoades et al., 2002), 47 of our predicted target sites fall in the open reading frames (ORFs). Five of the predicted target sites lie in the 3' UTR regions (At1g07650 [miR404], At1g12010 and At5g49870 [miR407a], At1g54870 [miR407b], and At2g47020 [miR408]). Interestingly, two miRNAs (miR399 and miR398) exhibit complementarity to the 5' UTR regions of their respective targets. miR399 appears to target four sites in the 5' UTR of At2g33770 (a putative ubiquitin conjugating enzyme E2) (Figure 4), whereas the miR398 family is likely to target the 5' UTR of At3g15640 (a member of cytochrome c oxidase).
We found that if more than one mRNA is predicted as the target of a miRNA, these targets tend to belong to the same gene family. For instance, miR400 is predicted to target more than 10 genes of the pentatricopeptide repeat (PPR) protein family; miR393 appears to target five genes belonging to the TIR1 (an F-box protein important for signaling by the phytohormone auxin) family; miR406 has the potential to target two members of spliceosome-related genes; miR397 appears to target two members of the laccase family; and miR389a.1 is capable of targeting four unknown proteins in the same family (Table 2). miR319c, a homolog of miR319/miRJAW, is likely to target the Teosinite branched1, cycloidea, and proliferating cell nuclear antigen factors family of transcription factors (Palatnik et al., 2003 ). miR399 family members are predicted to target the same gene, At2g33770 (ubiquitin conjugating enzyme E2). miRNAs that are represented by more than one member may recognize a consensus target sequence and, hence, might act on a common target or they might have tissue/stage-specific roles. In addition, different members of an miRNA family with slight changes in nucleotide sequence might also target different genes. For example, miR399b miRNA displays complementarity with a gene encoding a vesicle-associated membrane protein (At4g00170) in addition to the common target (At2g33770) of this family of miRNAs. miR397a is likely to target members of the laccase family, whereas another family member, miR397b, displays better complementarity with another gene (At3g60250, a casein kinase). Therefore, a slight change in miRNA sequence may alter its target preferences, distinguishing its role from that of other members of the same family.
The previously reported Arabidopsis miR161 is predicted to target PPR genes At1g06580(3), At1g62720(3), and At1g62670(3) (numbers in parenthesis are mismatches) (Rhoades et al., 2002 Some of the miRNA complementary sites are highly conserved in different members of the same gene family in Arabidopsis or in mRNA targets from different plant species (Table 3). For instance, miR393 complementary sites are highly conserved in F-box proteins in Arabidopsis, rice, and maize (Zea mays) (Table 3). miR397 is predicted to target laccases, and the target sites are conserved in Arabidopsis, rice, tobacco (Nicotiana tabacum), and Populus (Table 3). miR408 may target mRNAs encoding plantacyanins, and the complementary sites are conserved in rice, maize, chickpea (Cicer arietinum), and spinach (Spinacia oleracea) (Table 3). The complementary sites of miR389a.1 are conserved in mRNAs that encode proteins belonging to the same family (Table 3). The complementary sites are also conserved in spliceosomal proteins, which are predicted to be targeted by miR406. The conservation in the target sequence is observed not only at the amino acid level but also at the nucleotide level, including sequences at codon position 3 (Table 3). Furthermore, the sequence conservation in the target genes is markedly higher in the section corresponding to the 5' end of the miRNAs (Table 3).
Tissue and Developmental Expression Patterns of miRNAs Preferential expression of an miRNA in specific tissues or developmental stages may suggest a role in development for the miRNA. We investigated the tissue distribution of the newly identified miRNAs by RNA gel blot analysis on total RNA samples from various tissues of mature plants as well as young seedlings. We were able to detect a signal of the expected size for 13 miRNAs identified through cloning (Figure 5). The validation of other members of miR399, miR398, miR397, and predicted members of miR389 and miR407 were not attempted because of potential cross-hybridization. Some of the small RNAs were not detected on RNA gel blots but are still considered here as miRNAs because they were cloned and their precursors can adopt a hairpin structure. The miRNAs that could not be detected on RNA gel blots appeared only once in our library. A correlation was reported previously between the number of times a miRNA appeared in the library and its expression level. miRNAs identified in just one or a few clones have been difficult to detect on RNA gel blots, whereas those isolated many times are more easily detected (Grad et al., 2003 ; Lim et al., 2003; Kim et al., 2004). Some miRNAs may be expressed at low levels that are below the experimental detection threshold by RNA gel blot analysis, perhaps because of restricted tissue/cell-type expression (Grad et al., 2003; Lim et al., 2003; Kim et al., 2004).
Most of the previously known miRNAs in Arabidopsis are strongly expressed in floral tissues (Llave et al., 2002b However, other miRNAs in this study have quite divergent expression patterns. The most striking is that of miR398b, which could not be detected in the inflorescence (Figure 5A) and is preferentially expressed in leaves and young seedlings that consist mostly of young leaves. Similarly, miR399f, miR389b.3, and miR389a.1 are expressed more strongly in leaves and young seedlings, although they are also expressed in stems, roots, and inflorescence (Figures 5B to 5D). miR400 and miR397b show low but detectable expression in leaves, roots, and young seedlings but are undetectable in stems and inflorescence tissues (Figures 5H and 5I). miR403 and miR407 appear to be uniformly expressed in all tissues (Figures 5K and 5L). miR408 is strongly expressed in seedlings but weakly in all the tissues from adult plants (Figure 5M).
We also examined the expression of the miRNAs in dcl1-9 and hen1-1 mutant seedlings and compared the mutants to their wild-type background (Landsberg erecta ecotype). Consistent with the important roles of DCL1 (Reinhart et al., 2002
Abiotic Stress Regulation of miRNAs
Other Novel Endogenous Small RNAs Large censuses of small RNA cDNA libraries have revealed a significant class of other 21- to 22-nucleotide RNAs (Reinhart and Bartel, 2002 ; Ambros et al., 2003b; Aravin et al., 2003). These small RNAs differ from miRNAs in that they do not arise from hairpin precursors. Nevertheless, they display a strong size bias that suggests regulated processing by a Dicer-like activity. Furthermore, unlike some miRNAs, these small RNAs are generally not conserved phylogenetically. The recent discovery of a large class of tiny noncoding RNAs from C. elegans that share some features with miRNAs is an example for exceptional cases like this (Ambros et al., 2003b). These tiny noncoding RNAs are not even conserved within Caenorhabditis species (Ambros et al., 2003b). Another class of small RNAs, repeat-associated silencing RNAs, has been reported from Drosophila (Aravin et al., 2003). Repeat-associated silencing RNAs were noticed to be derived potentially from genomic regions rich in repeats (Aravin et al., 2003).
In this study, we have identified 102 endogenous small RNAs (putative siRNAs) that cannot be designated as miRNAs (Table 4). The putative siRNAs represent a heterogeneous class with respect to size and origin. The size ranged between 15 and 25 nucleotides, although the majority are in the range of 20 to 24 nucleotides. The small RNAs do not exhibit nucleotide preferences at their 5' end. The majority have a single locus in the Arabidopsis genome, although some (28) correspond to multiple loci (2 to 64). Twelve of the small RNAs corresponding to multiple loci appear to be derived from repeat-rich regions or centromeric regions (Table 4). Two small RNAs (P22-H04 and P43-A12) are derived from putative retrotransposons. The vast majority is found in intergenic regions, but some match to coding regions and introns in the antisense polarity. We consider those in the sense polarity as possible degradation products. Some of the putative siRNAs have perfect complementarity with transposases, LOB, AIG1, 23s rRNA, F-box protein, or pre-mRNA splicing factor. Some siRNAs display complementarity with their putative target genes with one to three mismatches. For instance, P78-C02 is complementary to At2g27152 (AAO3, a drought stress-inducible ABA biosynthesis enzyme) with two mismatches, whereas P52-B05 is complementary with a putative lipoxygenase with three mismatches. Some of these small RNAs may actually be miRNAs but are not considered as miRNAs at this time because the precursor sequences cannot adopt a fold-back structure using the mfold program. We were unable to predict targets for the majority of the putative siRNAs identified. These small RNAs might target promoter regions of protein coding genes for transcriptional gene silencing (Park et al., 2002
Small noncoding RNAs have recently emerged as important regulators of both transcriptional and posttranscriptional gene silencing. The identification of the entire set of small RNAs from an organism is of fundamental importance and would lay the foundation for understanding gene regulation involving small RNAs. The endeavor is as important as mining genes that code for proteins. The establishment of a comprehensive list of miRNAs from any organism will be instrumental for not only gene regulation studies but also for genome organization (e.g., transposon activity and chromatin assembly), phylogenetic comparison, comparative studies of development, and other evolutionary analysis. By sequencing a library of small RNAs from seedlings treated with different abiotic stresses, we are able to add 15 new families of miRNAs to the previously published 15 families in Arabidopsis. In addition, we identified new members of the previously reported miR171 and miR319 families (Table 1, Figure 1).
Some miRNAs are encoded by single copy genes, others by multiple genes. Most miRNA genes come from intergenic regions, but a sizable minority of miRNAs (about one-quarter of the human miRNA genes) occurs in the introns of protein coding genes, preferentially in the same orientation as the mRNAs (Bartel, 2004 Ten of the miRNAs that we have identified have clear orthologs or homologs in rice, Lotus, Medicago, or Populus, suggesting that they conduct important and perhaps conserved functions. On the other hand, many of the miRNAs are not conserved. These nonconserved miRNAs may play more species-specific roles.
Computational prediction of putative targets for miRNAs in plants has been facilitated by their perfect or near perfect complementarity to target genes (Rhoades et al., 2002
It was hypothesized that a complementary site in the coding region of an mRNA may lead to the cleavage of the target, whereas target sites located in 3' UTRs might attenuate translation (Bartel, 2004
Until now, no miRNA from animals or plants has been predicted to target the 5' UTR of a gene. In this work, we find that two miRNA families (miR399 and miR398) are likely to target 5' UTRs. miR398 family members are complementary to the 5' UTR of At3g15640 (cytochrome c oxidase). The miR399 has multiple complementary sites (four in Arabidopsis; Figure 4) within the 5' UTR of At2g33770 (putative ubiquitin conjugating enzyme E2). Future functional studies on the miR398 and miR399 families of miRNAs may reveal a novel mode of action of these miRNAs on their targets. The presence of multiple miRNA complementary sites is known in animal targets (Lee et al., 1993
The targeting of the 3' UTR of At2g47020 by miR408 is particularly interesting. The translation of At2g47020 mRNA may be repressed by miR408. At2g47020 is predicted to encode a peptide chain release factor important in mRNA translation. This suggests that the translational machinery may be targeted by an miRNA for translational regulation. The targeting of a ROS1-like DNA glycosylase and a SET domain protein by miR402 and miR407 miRNAs, respectively, is also very interesting. ROS1 is a DNA glycosylase/lyase required for the repression of siRNA-triggered transcriptional gene silencing by DNA demethylation (Gong et al., 2002
The concentration and activities of many cellular proteins are regulated by the ubiquitin pathway. Ubiquitination requires a cascade of three enzymatic activities for activating (E1), conjugating (E2), and ligating (E3) ubiquitin to a substrate. In this study, we have identified two miRNAs (miR399 and miR393) that may target components (ubiquitin conjugating enzyme E2 and ubiquitin ligase/TIR1) of the ubiquitination pathway. The result suggests a role for miRNAs in regulating the process of ubiquitination. Fungal laccases are well known for their roles in morphogenesis, pathogenicity, and lignin degradation (Mayer and Christopher, 2002
Several of the miRNAs discovered in this study are either upregulated or downregulated by abiotic stresses, suggesting that they may be involved in stress-responsive gene expression and stress adaptation. Consistent with this notion, the Arabidopsis hen1-1 and dcl1-9 mutants that are impaired in the production of some miRNAs are hypersensitive to abiotic stresses (our unpublished data). Stress-induced or upregulated miRNAs are expected to target negative regulators of stress responses or positive regulators of processes that are inhibited by stresses (e.g., cell division and expansion). On the other hand, stress downregulated miRNAs may repress the expression of positive regulators and/or stress upregulated genes. The significance of miR389a.1 miRNA downregulation by ABA and several stresses is unclear because this miRNA appears to target several genes encoding unknown proteins. The stress upregulation of miR393, which targets TIR1, suggests that stress may cause increased TIR1 mRNA degradation or translational repression. Because TIR1 is a positive regulator of auxin signaling by promoting the degradation of Aux/IAA proteins through ubiquitination (Dharmasiri and Estelle, 2002
The identification of endogenous siRNAs provides insights into the complexity of RNA-guided regulation. In this study, we have also uncovered a large number (102) of putative endogenous siRNAs that were not described previously, and these are distributed across all five chromosomes. We tested several of these small RNAs but were unsuccessful in detecting them on RNA gel blots, likely reflecting their low abundance. Unlike miRNAs, only a few of these small RNAs were represented by more than one clone in the sequenced library. These putative siRNAs are not conserved in other plants, which suggests species-specific endogenous RNA silencing mechanisms. The identification of unique intergenic regions as the sites of origin for most of the siRNAs indicated that they might be expressed independently. However, where multiple loci were identified for an siRNA, the origin was ambiguous. In plants, siRNAs have been suggested to mediate repression of retroelements via histone methylation as well as asymmetric DNA methylation (Zilberman et al., 2003 Several of the miRNAs reported in this study (miR402, miR403, miR397a, miR171b, and a predicted miRNA, miR389b.2) have also been identified by an NSF 2010 project, which can be accessed through the Arabidopsis Small RNA Project Web site of the Carrington lab (http://cgrb.orst.edu/smallRNA/). Despite the sequencing by the NSF 2010 project of several libraries from different genetic backgrounds and tissues, the overlap with ours is minimal. This suggests that the cloning of miRNAs in Arabidopsis is not saturated, and more miRNAs have yet to be identified. Therefore, continued efforts in the future are needed to identify the complete set of miRNAs and other small RNAs from Arabidopsis.
Cloning of miRNAs from Arabidopsis Total RNA was isolated from different samples (Arabidopsis thaliana ecotype Columbia; untreated, treated with cold stress 0°C for 24 h, 300 mM NaCl for 5 h, dehydration for 10 h, or 100-µM ABA for 3 h). Cloning of miRNAs was performed as described (Elbashir et al., 2001 ; Llave et al., 2002a). In brief, 600 µg of RNA was resolved through two lanes on a denaturing 15% polyacrylamide gel. Labeled DNA oligonucleotides were used as size standards. A gel fragment spanning the size range of 15 to 26 nucleotides was excised, and RNA was eluted overnight with 0.4 M NaCl at 4°C. The RNA was recovered by ethanol precipitation, dephosphorylated, and again ethanol precipitated. Then, the small RNAs were ligated sequentially to 5' and 3' RNA/DNA chimeric oligonucleotide adapters (Dharmacon Research, Boulder, CO). The 3' adapter oligonucleotide (5'-pUUUaaccgcatccttctcx-3'; uppercase, RNA; lowercase, DNA; p, phosphate; x, inverted deoxythymidine) (Elbashir et al., 2001) possessing a 5' monophosphate and a 3' inverted deoxythymidine to prevent self ligation, was then ligated to the dephosphorylated small RNA. The ligation product was recovered from the gel, 5' phosphorylated, and the RNA was recovered by ethanol precipitation. Next, the 5' adapter (5'tactaatacgactcactAAA; uppercase, RNA; lowercase, DNA) was ligated to the phosphorylated ligation product as described above. The ligation products from the second ligation reaction were excised and eluted from the gel. Reverse transcription reaction was performed using the RT primer (5'-TTTTCTGCAGAAGGATGCGGTTAAA-3'; bold, PstI site). This was followed by PCR using the reverse (RT primer) and forward (5'-AAACCATGGTACTAATACGACTCACTAAA-3'; bold, NcoI site) primers. The PCR product was purified by phenol/chloroform extraction, ethanol precipitated, and digested with PstI and NcoI. After additional phenol/chloroform extraction and ethanol precipitation, the digested products were ligated to a PstI-NcoI–linearized pGEM-T Easy vector using T4 DNA ligase. The inserts from individual colonies were amplified by T7 and SP6 primers and sequenced. The sequences were subsequently processed and used for BLAST analysis against the Arabidopsis sequences in The Arabidopsis Information Resource database (http://www.arabidopsis.org/) and other plant sequences in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST/).
Prediction of Fold-Back Structures
RNA Gel Blot Analysis
We thank Xuemei Chen for providing hen1-1 and dcl1-9 seeds and Becky Stevenson for technical assistance during the course of this work. We also thank Shou-Wei Ding and Andre Jaggendorf for critical reading of the manuscript. We thank Anastasia Khavorova and Annaleen Vermeulen for their help in analyzing thermodynamic profiles of miRNAs. This work was supported by National Institutes of Health Grant R01GM0707501 and National Science Foundation Grant IBN-9808398 to J.K.Z.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the Instructions for Authors (www.plantcell.org) is: Jian-Kang Zhu (jian-kang.zhu{at}ucr.edu).
Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.022830. Received March 22, 2004; accepted June 3, 2004.
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ABSTRACT
INTRODUCTION
-32P-ATP using T4 polynucleotide kinase (Invitrogen). Blots were prehybridized for at least 1 h and hybridized overnight using PerfectHYB Plus buffer (Sigma, St. Louis, MO) at 38°C. Blots were washed three times (two times with 2x SSC [1x SSC is 0.15 M NaCl and 0.015 M sodium citrate] and 0.1% SDS for 20 min and once with 1x SSC and 0.1% SDS for 20 min) at 50°C. The membranes were briefly air dried and then exposed to a phosphorimager or autoradiographed. 