Genetic and Molecular Approaches to Assess MicroRNA Function
Recent advancements in sequencing small RNA populations have enabled the discovery of a multitude of microRNAs (miRNAs) in numerous plant species. Moreover, bioinformatic analyses to predict, or molecular approaches such as 5′-RACE or degradome analyses to validate miRNA targets, have elucidated the tremendous scope of miRNA regulation. The challenge now lies in determining the functional significance of these miRNA:target regulatory relationships. However, MIRNA loss-of-function mutants have been scarce, in part due to the existence of multiple functionally redundant miRNA family members. Consequently, much of the functional analysis of miRNAs has derived from gain-of-function approaches, including activation tagging of miRNAs, the identification of miRNA binding site mutations and transgenic approaches such as over-expressing miRNAs or miRNA-resistant targets. Here, we review the above molecular and genetic methodologies and assess their pros and cons in determining function. We compare the conclusions drawn from these approaches to those of the few known loss-of-function analyses, using analysis of the miR159 system in Arabidopsis as an example, where the loss-of-function mutants find a much narrower functional target specificity than what is insinuated from other approaches.
KeywordsmiRNA Target MIRNA Gene miRNA Regulation Plant miRNAs miRNA Binding Site
RSA and AAM gratefully acknowledge the support of Australian Research Council Discovery Grants DP1097150 and DP0773270, respectively.
- Alonso-Peral MM, Li J, Li Y, Allen RS, Schnippenkoetter W, Ohms S, White RL, Millar AA (2010) The miR159 regulated GAMYB-like genes inhibit growth and promote programmed cell death in Arabidopsis. Plant Physiol 110:160–30Google Scholar
- Jones-Rhoades, M.W., Bartel, D.P., and Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology 57:19–53Google Scholar
- Jordan SD, Kruger M, Willmes DM, Redemann N, Wunderlich FT, Bronneke HS, Merkwirth C, Kashkar H, Olkkonen VM, Bottger T et al (2011) Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism. Nat Cell Biol 13:434–446PubMedCrossRefGoogle Scholar
- Reyes JL, and Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination Plant J 49:592–606Google Scholar
- Swaminathan K, Alabady MS, Varala K, De Paoli E, Ho I, Rokhsar DS, Arumuganathan AK, Ming R, Green PJ, Meyers BC et al (2010) Genomic and small RNA sequencing of Miscanthus × giganteus shows the utility of sorghum as a reference genome sequence for Andropogoneae grasses. Genome Biol 11:R12PubMedCrossRefGoogle Scholar