Cloning and Characterization of MicroRNAs from Rice

Abstract

MicroRNAs (miRNAs) are a growing family of small noncoding RNAs that downregulate gene expression in a sequencespecific 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.

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

“…MiRNAs can be identified by direct cloning (Ramachandra et al, 2008;Sunkar et al, 2005), high-throughput sequencing (Chi et al, 2011;Soares et al, 2009) and computational homology-based methods (Jones-Rhoades and Bartel, 2004;Li et al, 2010). And recently, a lot of miRNAs have been identified by high-throughput sequencing in various animals and plants for its high performance, including rainbow trout (Ma et al, 2012).…”

Identification of Potential microRNAs and Their Targets in Brassica rapa L.

“…These 2 methods have been used to identify hundreds of miRNAs in several plant species, including the model plant Arabidopsis thaliana (AlvesJunior et al, 2009;Buhtz et al, 2008;Jagadeeswaran et al, 2009;Jin et al, 2008;Klevebring et al, 2009;Song et al, 2009;Sunkar et al, 2005;2008;Szittya et al, 2008;Xie et al, 2007;Zhang et al, 2006b;2007a;2008). Recently, many miRNAs have been shown to regulate a wide range of biological functions of plants.…”

Molecular evolution and selection of a gene encoding two tandem microRNAs in rice

“…To date, 4584 miRNAs have been identified in animals and plants (miRBase, http://microrna.sanger.ac.uk/, Release 9.2). In rice, 242 miRNAs of 62 families have been identified by cloning or computational prediction [6][7][8][9][10].…”