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MiR156 biogenesis is involved in the response to ambient temperature changes in plants W. Kim and J. H. Ahn Creative Research Initiative, School of Life Sciences and Biotechnology, Korea University, South Korea ([email protected]) Plant microRNAs (miRNAs) are small non-coding RNA playing an important regulatory role in plant development. It was recently reported that a subset of miRNAs and their target gene are involved in the response to ambient temperature changes. However, ambient temperature sensing mechanism of plant miRNAs is not known. In this study, mature and precursor expression of miR156a and c, which are considered as major product of miR156, were anticorrelated at different ambient temperatures. RLM 5`-RACE assay revealed that more cleavage products of miR156a and miR156c precursors were produced at low temperature, suggesting that increased and decreased expression of miR156 mature and precursor form, respectively, were caused by enhanced cleavage of miR156 precursor at low temperature. Mapping of the cleavage site of miR156 precursor by sequencing of cleavage fragments indicated that major cut of miR156a occurred at 3` overhang below miR156/miR156* duplex. This is consistent with previous report demonstrating that many plant miRNA precursors are processed in a canonical base-to-loop mode. However, in the case of miR156c, it is likely that miR156c follows loop-to-base processing. The expression level of genes impaired in miRNA biogenesis was not altered at different ambient temperatures, indicating that anti-correlation between mature and precursor form was not mediated by transcript level of miRNA biogenesis genes. Based on comparison of expression analysis of ambient temperatureresponsive miRNA (ATRM) precursor with their mature expression at different ambient temperatures, we could hypothesize that ATRM is divided into two groups, being regulated ‘at transcriptional level’ and ‘at biogenesis level’. Taken together, our results suggest the involvement of miR156 biogenesis in the response to ambient temperature changes in plants.