Detection of MicroRNA Processing Intermediates Through RNA Ligation Approaches
MicroRNAs (miRNA) are small RNAs of 20–22 nt that regulate diverse biological pathways through the modulation of gene expression. miRNAs recognize target RNAs by base complementarity and guide them to degradation or translational arrest. They are transcribed as longer precursors with extensive secondary structures. In plants, these precursors are processed by a complex harboring DICER-LIKE1 (DCL1), which cuts on the precursor stem region to release the mature miRNA together with the miRNA*. In both plants and animals, the miRNA precursors contain spatial clues that determine the position of the miRNA along their sequences. DCL1 is assisted by several proteins, such as the double-stranded RNA binding protein, HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). The precise biogenesis of miRNAs is of utter importance since it determines the exact nucleotide sequence of the mature small RNAs and therefore the identity of the target genes. miRNA processing itself can be regulated and therefore can determine the final small RNA levels and activity. Here, we describe methods to analyze miRNA processing intermediates in plants. These approaches can be used in wild-type or mutant plants, as well as in plants grown under different conditions, allowing a molecular characterization of the miRNA biogenesis from the RNA precursor perspective.
Key wordsMicroRNA Processing Precursor RNA ligation NGS Plants Arabidopsis
Supported by grants of Argentinean Ministry of Science, PICT-2015-3557 and PICT-2016-0761 to J.P. CONICET fellowships to B.M. and A.M.L.R.
- 9.Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhu JK (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190CrossRefGoogle Scholar
- 38.Moro B, Chorostecki U, Arikit S, Suarez IP, Höbartner C, Rasia RM, Meyers BC, Palatnik JF (2018) Efficiency and precision of microRNA biogenesis modes in plants. Nucleic acids research 46(20):10709–10723. https://doi.org/10.1093/nar/gky853