MicroRNAs in plants

Abstract

MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.

Figure 1

Fold-back secondary structures of Arabidopsis miRNA predicted precursors as determined by the RNAfold program. The miRNA sequences are in red. For miR156 and miR169, RNAs from the other side of the fold-back (boxed in blue) were each cloned once. The duplexes that could form between these RNAs and the miRNA from the other strand have ∼2-nt 3′ overhangs characteristic of Dicer cleavage (Elbashir et al. 2001).

Figure 2

Developmental expression of Arabidopsis miRNAs. Total RNA from Columbia seedlings (Se), leaves (L), stems (St), flowers (F), and siliques (Si) was analyzed on Northern blots by hybridization to end-labeled DNA oligonucleotide probes complementary to the miRNA. The lengths of end-labeled RNA oligonucleotides run as a size marker (M) are noted to the left of each panel. Although miR165 and miR166 sequences and miR170 and miR171 sequences are too closely related to be reliably distinguished by hybridization probes, miR156 and miR157 should be specifically recognized (Lau et al. 2001), as reflected in their different levels of expression in seedlings and siliques. miR159 and miR164 show a similar expression profile to miR165, whereas miR160, miR162, and miR168 have similar profiles to miR158 (data not shown). The low expression level of most miRNAs in leaves and siliques might reflect a difference in the efficiency of small RNA recovery with the RNA isolation method used for these two tissues (see Materials and Methods). Blots were stripped and reprobed with an oligonucleotide probe complementary to U6 as a loading control.

Figure 3

Expression of miR169 is dependent on CARPEL FACTORY. Total RNA from wild-type Landsberg erecta (CAF/CAF), heterozygous (CAF/caf), and homozygous (caf/caf) carpel factory leaves (L), stems (St), and flowers (F) was analyzed on a Northern blot. RNA size markers (M) are noted to the left. The blot probed for miR158 was stripped and reprobed with a U6 end-labeled DNA probe as a loading control.

Figure 4

Conservation between the Arabidopsis and Oryza predicted stem–loop precursors. (A) miR162 homologs. (B) miR164 homologs. Sequence homology is seen within the miRNA (in red), its paired sequences, and a few base pairs adjacent to the miRNA. The remainder of the sequence has drifted considerably, with the main constraint being the formation of a stem–loop structure.

Figure 5

A cluster of small RNAs derived from Chromosome 2. Arrows represent the two predicted genes in this region, and vertical lines represent the genomic positions of the six cloned RNAs. Sequences of the RNAs are listed, with cloning frequencies in parentheses.