MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of protein coding genes. Although the biogenesis and function of miRNAs are well characterised, we know very little about their turn-over. We have developed a system in Arabidopsis plants where we can monitor the turn-over of a specific miRNA by the expression level of a reporter GFP gene. The miRNA is induced by low level of sulphate (1,2) and because the reporter GFP mRNA contains an engineered target site, plants grown on low sulphur media do not express GFP. Expression of the miRNA can be switched off by supplementing the media with sulphur and in about 2-3 days GFP expression is visible because no new miRNAs are produced. Seeds were mutagenized and by next autumn we will have the M2 population that can be screened for plants that express GFP either earlier or later than the wild type plants. Plants that start expressing GFP slower than wild type plants are expected to have mutations in genes required for turn-over of miRNAs because if turn-over is deficient it takes longer for the miRNAs to decay. Plants that start expressing GFP quicker than wild type plants are expected to have mutations that suppress turn-over of miRNAs. It is possible that mutations will happen in the GFP gene itself or in the sulphur pathway that regulates the expression of the miRNA but these can be distinguished from the real turn-over mutants by directly monitoring the level of the miRNA by Northern blot analysis. Therefore the high throughput screen will be based on GFP expression and then a small number of putative mutants will be tested by Northern blot analysis. Once the screen is completed, the mutations will be identified by either classical map-based cloning (3) or by next generation sequencing (4). Finally the mutants will be characterised to investigate the mechanism of miRNA turn-over.  

(i) Kawashima, CG., Yoshimoto, N., Maruyama-Nakashita, A., Tsuchiya, Y., Saito, K., Takahashi, H. and Dalmay, T. (2009) Sulphur starvation induces the expression of microRNA-395 and one of its target genes but in different cell types. Plant Journal, 57(2):313-21

(ii) Kawashima, C., Matthewman, C.,Huang, S.,Lee, BR., Yoshimoto, N., Koprivova, A., Rubio-Somoza, I., Todesco, M., Rathjen, T., Saito, K., Takahashi, H., #Dalmay, T., #Kopriva, S. (2011) Interplay of SLIM1 and miR395 in regulation of sulfate assimilation in Arabidopsis. Plant Journal, 66(5):863-76

(iii) Hernandez-Pinzon, I, Yelina, NE, Scwach, F, Studholme, D, Baulcombe, D and Dalmay T (2007) SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA. The Plant Journal, 50(1):140-8.

(iv) Galvão VC, Nordström KJ, Lanz C, Sulz P, Mathieu J, Posé D, Schmid M, Weigel D, Schneeberger K. (2012) Synteny-based mapping-by-sequencing enabled by targeted enrichment. Plant J. 2012 Aug;71(3):517-26