The role of methylation and uridylation in microRNA metabolism in Arabidopsis

Once large amounts ofRDR2 and RDR6 are available, we will perform kinetic studies on their enzymatic activities. First, in vitro transcribed RNA templates with or without a 5' cap and with or without a polyA tail will be incubated with the RDR proteins in the presence of radioactive UTP to examine their primer independent activity (if they have such activities). Products will be visualized by gel electrophoresis followed by autoradiography. The Kd for each type of substrate will be determined. This analysis will help us understand what RNAs tend to serve as RdRP templates. Next, we will examine the primer-dependent activity, if any, of the two proteins. An unmethylated RNA oligo fully complementary to a region of the template will be added. The position of the RNA oligo needs to be experimentally determined so that the product of the primer-dependent activity will be different in size from that of the primer-independent activity to allow the evaluation of the primer-dependent activity. Finally, once the assay has been worked out, we will determine the kinetics of the primer-dependent reactions when either the methylated or the unmethylated primer is used.