Cloning and characterization of microRNAs from rice

Abstract

MicroRNAs (miRNAs) are a growing family of small noncoding RNAs that downregulate gene expression in a sequence-specific manner. The identification of the entire set of miRNAs from a model organism is a critical step toward understanding miRNA-guided gene regulation. Rice (Oryza sativa) and Arabidopsis thaliana, two plant model species with fully sequenced genomes, are representatives of monocotyledonous and dicotyledonous flowering plants, respectively. Thus far, experimental identification of miRNAs in plants has been confined to Arabidopsis. Computational analysis based on conservation with known miRNAs from Arabidopsis has predicted 20 families of miRNAs in rice. To identify miRNAs that are difficult to predict in silico or not conserved in Arabidopsis, we generated three cDNA libraries of small RNAs from rice shoot, root, and inflorescence tissues. We identified 35 miRNAs, of which 14 are new, and these define 13 new families. Thirteen of the new miRNAs are not conserved in Arabidopsis. Four of the new miRNAs are conserved in related monocot species but not in Arabidopsis, which suggests that these may have evolved after the divergence of monocots and dicots. The remaining nine new miRNAs appear to be absent in the known sequences of other plant species. Most of the rice miRNAs are expressed ubiquitously in all tissues examined, whereas a few display tissue-specific expression. We predicted 46 genes as targets of the new rice miRNAs: 16 of these predicted targets encode transcription factors, and other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of miRNA-mediated mRNA cleavage. Our identification of new miRNAs in rice suggests that these miRNAs may have evolved independently in rice or been lost in other species.

Figure 1.

Size Distribution of New miRNAs Cloned from Rice.

Figure 2.

OsmiR396d Is a Unique Member of the Previously Predicted OsmiR396 Family in Rice. Sequence alignment of miR396d and predicted members of OsmiR396 family. Bold letter represents the additional nucleotide (nt) in the new miRNA.

Figure 3.

Schematic Representation of the Biogenesis of OsmiR444. The hairpin structure requires parts of exon 2 and exon 3 of the host transcript J033125N22.

Figure 4.

The miR444 Family Is Conserved in Monocots. (A) Alignment of miR444 sequence from rice with the predicted homologs in wheat, barley, maize, sorghum, and sugarcane. (B) Predicted fold-back structures of miR444 precursors from rice, wheat, barley, maize, sorghum, and sugarcane.

Figure 5.

Predicted Fold-Back Structure of OsmiR436 Precursor. The fold-back structure was predicted with use of a 720-bp processed transcript. Protruding stem-loops in the 3′ arm of the hairpin are indicated by the slashes (\).

Figure 6.

Expression Patterns of miRNAs Cloned from Rice. RNA gel blots of total RNA isolated from different tissues were probed with labeled oligonucleotides. The blots also included RNA from maize and Arabidopsis. The tRNA and 5S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. Labeled RNA oligonucleotide was used as a size marker, and the position was indicated. nt, nucleotides.

Figure 7.

Expression Patterns of New Rice miRNAs That Are Conserved in Another Monocot (Maize) or in Dicot (Arabidopsis) and Monocot (Maize). The tRNA and 5S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. Labeled RNA oligonucleotide was used as a size marker, and the position was indicated. nt, nucleotides.

Figure 8.

OsmiR439 Is Predicted to Target Three Sites within the ORF of Its Target Gene, 11667.m02576. Numbers represent the position of target sites in the ORF. Amino acid sequence corresponding to the target nucleotide sequence is shown.

Figure 9.

Identification of miRNA-Guided Cleavage Products of Target Genes in Rice. (A) mRNA 7448.m00137. (B) mRNA 11668.m00935. (C) mRNA 11668.m04852. (D) mRNA 11686.m04227. Mapping of cleavage sites was done by RNA ligase-mediated 5′ RACE. Partial mRNA sequences from target genes were aligned with miRNAs. Numbers indicate the fraction of cloned PCR products terminating at different positions.