Background. The plant miRNAs represent an important class of endogenous small RNAs that guide cleavage of an mRNA target or repress its translation to control development and adaptation to stresses. MiRNAs are nuclear-encoded genes transcribed by RNA polymerase II, producing a primary precursor that is subsequently processed by DCL1 an RNase III Dicer-like protein. In rice hundreds of miRNAs have been described or predicted, but little is known on their genes and precursors which are important criteria to distinguish them from siRNAs. Here we develop a combination of experimental approaches to detect novel miRNAs in rice, identify their precursor transcripts and genes and predict or validate their mRNA targets. Results. We produced four cDNA libraries from small RNA fractions extracted from distinct rice tissues. By in silico analysis we selected 6 potential novel miRNAs, and confirmed that their expression requires OsDCL1. We predicted their targets and used 5'RACE to validate cleavage for three of them, targeting a PPR, an SPX domain protein and a GT-like transcription factor respectively. In addition, we identified precursor transcripts for the 6 miRNAs expressed in rice, showing that these precursors can be efficiently processed using a transient expression assay in transfected Nicotiana benthamiana leaves. Most interestingly, we describe two precursors producing tandem miRNAs, but in distinct arrays. We focus on one of them encoding osa-miR159a.2, a novel miRNA produced from the same stem-loop structure encoding the conserved osa-miR159a.1. We show that this dual osa-miR159a.2-osa-miR159a.1 structure is conserved in distant rice species and maize. Finally we show that the predicted mRNA target of osa-miR159a.2 encoding a GT-like transcription factor is cleaved in vivo at the expected site. Conclusion. The combination of approaches developed here identified six novel miRNAs expressed in rice which can be clearly distinguished from siRNAs. Importantly, we show that two miRNAs can be produced from a single precursor, either from tandem stem-loops or tandemly arrayed in a single stem-loop. This suggests that processing of these precursors could be an important regulatory step to produce one or more functional miRNAs in plants and perhaps coordinate cleavage of distinct targets in the same plant tissue.
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