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Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis - PubMed

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Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis

Ramanjulu Sunkar et al. Plant Cell. 2004 Aug.

Abstract

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.

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Figures

Figure 1.
Figure 1.

miRNA Families in Arabidopsis and Other Plants (Rice, Lotus, and Medicago). miRNAs cloned in this study are labeled in pink. The letter P following miRNA loci (miR399, miR389b.2, and miR407b) denotes predicted miRNAs based on similarity with the cloned sequences and the ability of their precursor sequences to form hairpin structures. miR399 family is represented by six loci in the Arabidopsis (A.t.) genome. Only five loci (a, b, c, d, and e) are shown in Figure 1A because the locus d is represented twice with identical sequence (Table 1). Identical and conserved nucleotides are labeled in red. Blue letters represent changes in nucleotide sequences in some family members. The location of the predicted Arabidopsis miRNAs in the chromosomes is shown to the right of the sequence. Sequences were aligned with the ClustalW program (Thompson et al., 1994). All miRNAs from rice (O.s.), Lotus, and Medicago are from computer prediction only.

Figure 2.
Figure 2.

Conserved Genomic Organization of the miR399 miRNA Cluster in Arabidopsis and Rice. miR399 loci d, e, and f are located within a 2.0-kb region on chromosome 2 in Arabidopsis, and part of this cluster is conserved in rice (chromosome 5). The hairpin precursor is indicated as a box, and the position of the miRNA in the precursor is shown as thick vertical lines. Arrow on top of the box indicates the orientation of miRNAs.

Figure 3.
Figure 3.

Predicted Fold-Back Structures of miR389a.1 Precursors with Flanking Sequences on Chromosome 1 and Chromosome 2. (A) Chromosome 1. (B) Chromosome 2. The fold-back structure shown in (B) is expected to yield two miRNAs, one each on 3′ and 5′ arms. The cloned miRNA sequence is labeled in red, and the predicted one is labeled in blue.

Figure 4.
Figure 4.

Schematic Representation of the At2g33770 mRNA Showing the 5′ UTR, ORF, and 3′ UTR Regions. Predicted miRNA targeting sites in the 5′ UTR region are shown (black rectangles). The enlarged portion shows the pairing between miR399f and the target sites.

Figure 5.
Figure 5.

Expression Patterns of miRNAs Cloned from Arabidopsis. RNA gel blots of total RNA isolated from different tissues and 2-week-old seedlings were probed with labeled oligonucleotides. The blots also included RNA from dcl1-9 and hen1-1 mutants and their wild type, Landsberg erecta. The samples are leaf (L), stem (S), root (R), inflorescence (I), 2-week-old-seedlings (Se), Landsberg erecta (Ler), dcl1-9 (dcl), and hen1-1 (hen). The tRNA and 5S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and serve as loading controls.

Figure 6.
Figure 6.

Regulation of miRNA Expression by Abiotic Stresses. Shown are RNA gel blots of total RNA isolated from 2-week-old seedlings untreated or treated with different abiotic stresses. The tRNA and 5S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and serve as loading controls.

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