Sequence requirements for efficient activity of the microRNA miR398 in Arabidopsis thaliana

MARTIN, ANA PAULA; LODEYRO, ANABELLA F.; CARRILLO, NÉSTOR; PALATNIK, JAVIER F.

La Plata

Congreso; XXVIII Reunión Argentina de Fisiología Vegetal; 2010

SOCIEDAD ARGENTINA DE FISIOLOGÍA VEGETAL (SAFV)

MicroRNAs (miRNAs) are a class of regulatory RNAs of ~21 nucleotides that post-transcriptionally regulate eukaryotic gene expression by directing mRNA cleavage or inhibiting translation. They are involved in diverse physiological processes like development, differentiation and metabolism. The miRNA miR398 regulates Cu/Zn superoxide dismutases (CSD1 and CSD2) that detoxify superoxide radicals; and a subunit of Cytochrome C Oxidase, COX5b1. All these proteins bind Cu as a cofactor. Copper deficiency in the growth media induces miR398, which in turn guides the degradation of its targets. As a consequence, the available copper is directed to essential proteins. It has been observed that miR398 exhibits differential activity towards its targets, being CSD1 and CSD2 more sensitive than COX5b1 to the miRNA levels. Interestingly, each gene regulated by miR398 has a specific nucleotide sequence at the target site that has been conserved during evolution. One possibility is that these differences in the target sites underlie the differential activity of miR398 towards its targets. To elucidate this point we prepared several variants of CSD2 and COX5b1 with specific mutations at the target site and expressed them fused to GFP under the control of the constitutive promoter 35S. Then, we analyzed the expression levels of each transgene under different Cu conditions. These experiments allowed us to compare the sensitivity of the different target sites to miR398. A model showing the relative importance of the target site sequence on miR398 activity will be presented.