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Plant Physiology 146:3-4 (2008) © 2008 American Society of Plant Biologists Arabidopsis MicroRNAs
University of Illinois
MicroRNAs (miRNAs) are noncoding RNAs approximately 21 nucleotides long that regulate gene expression. This regulation is achieved in two ways: binding to specific target RNAs targeting them for degradation or possibly attenuating translation of the target mRNA. Unlike small interfering RNAs (siRNAs) that are derived from mRNAs, transposons, viruses, or heterochromatin DNA, miRNAs are encoded by distinct genomic loci and are most likely transcribed by RNA polymerase II (pol II). After transcription, the precursor miRNA folds back on itself, forming an imperfect stem loop precursor structure that is cleaved by a member of the Dicer family, double-stranded RNA-specific nucleases.
In plants miRNAs are primarily involved in regulation of growth and development, although they also have been shown to be involved in the regulation of plant response to nutrient stresses (for review, see Bartel and Bartel, 2003
Binding of the miRNA to its target is imperfect and this has made identification difficult. The majority of known miRNAs have been identified by cloning and sequencing small RNAs. A disadvantage of this is the presence of other small RNAs such as siRNAs; the article discussed in this month's High Impact, by Xie et al. (2005)
To identify novel plant miRNAs, Xie et al. (2005)  constructed several small RNA libraries. However, siRNAs are of about the same size and thus could interfere with the isolation of miRNAs. To enrich for miRNAs, the researchers took advantage of two Arabidopsis (Arabidopsis thaliana) mutants with low levels of siRNAs due to lesions in key enzymes in the siRNA synthesis pathway. These mutants had normal levels of miRNAs and yielded libraries with a 2.2-fold increase in miRNAs relative to wild-type libraries. To identify new miRNAs from the cloned libraries, a series of six computational filters were created based on a founder set of validated Arabidopsis miRNAs, as well as miRNA consensus properties from both plants and animals. This ultimately yielded 18 small RNAs from 13 loci as possible new miRNAs. MiRNA accumulation from the candidates was determined in wild type, as well as in mutant plants with miRNA and siRNA defects. Of these 13 candidates, four (miR390, miR391, miR403, and miR447) were determined to be genuine miRNAs. The targets for three of these have been predicted and validated (Allen et al., 2005), increasing the number of experimentally validated Arabidopsis miRNA genes to 99 representing 25 families. It is likely that in plants miRNA is transcribed by RNA pol II similar to what is found in animal systems. In order for transcription by RNA pol II to occur, RNA needs to contain a 5' cap. To determine if RNA pol II also transcribes miRNA in plants, poly(A+) RNA was treated with calf intestine phosphatase plus tobacco acid pyrophosphatase to select for 5' ends that have a 5' cap. The treated RNA was then used for RNA ligase-mediated 5' RACE using locus-specific primers. Of the 99 validated genes, amplification was successful for 63 of the miRNA primary transcripts representing 52 loci (53%), strongly suggesting that some plant miRNAs are transcribed by RNA pol II. The majority (79%) of the 5'RACE products obtained were of uniform size. Those that were not fell into two groups having either two or three distinct size classes. The authors suggested that a possible reason for this could be due to different transcription start sites among the genes associated with a given loci. The PCR fragments of the genes from the 52 loci were cloned and sequenced to further examine the 5'-untranslated regions of the miRNAs and determine if this were the case. Of the majority of the different size classes, multiple transcriptions start sites were indeed found. The sequencing also found that the majority of transcripts were initiated with adenosine followed by a pyrimidine, again consistent with RNA pol II transcription. As mentioned above, only 53% of the tested miRNA loci produced a product with 5'RACE. This could be due to primers being located within introns, or the miRNA not being expressed in the tissue tested or just not expressed. To investigate this further, an informatics approach was taken and the Arabidopsis Small RNA Project database was scanned for sequences corresponding to the miRNAs that gave negative results. Eighteen were identified in either that database or in another independent small RNA library. Locus-specific primers downstream of the predicted precursor foldback sequence were designed for the remaining predicted miRNA genes to be used for 3'RACE. All together, the expression of 73 of the 99 Arabidopsis miRNA is supported by the data presented in this work.
Not much is known about the regulation of miRNA expression. It has been shown that miR162 regulates expression by targeting DCL1, the Dicer family member responsible for cleaving pre-miRNA (Xie et al., 2003 ). Knowing the promoter elements present in miRNAs would greatly help in understanding how expression of the genes is regulated. As part of their study, Xie et al. (2005) sequenced the 5' end of known miRNAs and found the TATA box sequence motif in their promoter region. A study by Megraw et al. (2006) has expanded on this and identified four other transcription factor binding motifs overrepresented in Arabidopsis miRNAs. Using the 63 5'RACE miRNA sequences from the Xie et al. (2005) study, Megraw et al. (2006) analyzed up to 800 nucleotides further upstream to identify known transcription factor binding elements present in this region. They found that AtMYC2, ARF, SORLREP3, and LFY were overrepresented in the miRNAs relative to protein-coding gene promoters and randomly sampled genomic sequences, suggesting the involvement of these transcription factors in miRNA transcription. The LFY gene has been shown to play a key role in flower development and thus miRNA regulation by LFY would be fitting.
Information on Arabidopsis miRNAs from the Xie et al. (2005)
Many studies are ongoing to validate the miRNAs that have been identified, as well as the identification of other new candidate miRNAs and the determination of how conserved miRNAs are between plants. That many miRNAs are found across plant species (Willmann and Poethig, 2007 ) suggests an ancient origin of these important regulatory units.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.900244.
Allen E, Xie Z, Gustafson AM, Carrington JC (2005) MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121: 207–221[CrossRef][ISI][Medline] Bartel B, Bartel DP (2003) MicroRNAs: at the root of plant development? Plant Physiol 132: 709–717 Chiou TJ (2007) The role of microRNAs in sensing nutrient stress. Plant Cell Environ 30: 323–332[CrossRef][Medline] Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CL (2006) Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell 18: 412–421 Lu C, Tej SS, Luo SJ, Haudenschild CD, Meyers BC, Green PJ (2005) Elucidation of the small RNA component of the transcriptome. Science 309: 1567–1569 Megraw M, Baev V, Rusinov V, Jensen ST, Kalantidis K, Hatzigeorgiou AG (2006) MicroRNA promoter element discovery in Arabidopsis. RNA 12: 1612–1619 Meyers BC, Souret FF, Lu C, Green PJ (2006) Sweating the small stuff: microRNA discovery in plants. Curr Opin Biotechnol 17: 139–146[ISI][Medline] Talmor-Neiman M, Stav R, Frank W, Voss B, Arazi T (2006) Novel micro-RNAs and intermediates of micro-RNA biogenesis from moss. Plant J 47: 25–37[CrossRef][ISI][Medline] Tesfaye M, Liu JQ, Allan DL, Vance CP (2007) Genomic and genetic control of phosphate stress in legumes. Plant Physiol 144: 594–603 Willmann MR, Poethig RS (2007) Conservation and evolution of miRNA regulatory programs in plant development. Curr Opin Plant Biol 10: 503–511[CrossRef][ISI][Medline] Xie ZX, Kasschau KD, Carrington JC (2003) Negative feedback regulation of Dicer-Like1 in Arabidopsis by microRNA-guided mRNA degradation. Curr Biol 13: 784–789[CrossRef][ISI][Medline] Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138: 2145–2154
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