Identification of novel maize miRNAs by measuring the precision of precursor processing

To find new miRNA in this important crop, small RNAs from mixed tissues were sequenced, resulting in over 15 million unique sequences. Our sequencing effort validated 23 of the 28 known maize miRNA families, including 49 unique miRNAs. Using a newly established criterion, based on the precision of miRNA processing from precursors, we identified 66 novel miRNAs in maize. These miRNAs can be grouped into 58 families, 54 of which have not been identified in any other species. Five new miRNAs were validated by northern blot. Moreover, we found targets for 23 of the 66 new miRNAs. The targets of two of these newly identified miRNAs were confirmed by 5'RACE.We have implemented a novel method of identifying miRNA by measuring the precision of miRNA processing from precursors. Using this method, 66 novel miRNAs and 50 potential miRNAs have been identified in maize.MiRNAs are known to play crucial roles in the regulation of gene expression in plants [1], including functions such as, leaf polarity, auxin response, floral identity, flowering time, and stress response [2-7]. MiRNAs are typically ~21 nucleotides in length. In plants, miRNA genes are transcribed by RNA polymerasell into primary miRNA transcripts (pri-miRNA) which can form imperfect stem-loop secondary structure [8,9]. Then the pri-miRNAs are trimmed and spliced into miRNA/miRNA* duplex by Dicer-like1 (DCL1) with the help of dsRNA binding protein HYL1 and dsRNA methylase HEN1 [1,10-12]. The length of the pre-miRNAs in plants ranges from about 80-nt to 300-nt, and is more variable than in animals. After being transported to the cytoplasm, the mature miRNAs can match to the corresponding target mRNAs through RNA-induced silencing complex (RISC) and the miRNA* are thought to be degraded [1,13]. MiRNAs regulate their target mRNA either by cleaving in the middle of their binding sites or by translational repression [14,15]. The plant miRNAs are highly complementary to their targets with about 0~4 nucleotides mismatches