Nucleotidyl transferase assays for URT1.

<p>Recombinant His-tagged wild type URT1 or a catalytic mutant was included in the reactions. The wild type and mutant proteins are indicated by “W” and “M”, respectively. The proteins were incubated with an RNA oligonucleotide (oligo) in the presence of a nucleotide triphosphate, and the products were resolved on 15% denaturing polyacrylamide gels. miR173 and miR173-2’-Me are un-methylated and methylated, respectively, on the 2’ OH of the 3’ terminal ribose. Nucleotidyl transferase activity is represented by the presence of higher molecular weight bands relative to the input miRNA. Note that in lane 4, the substrate RNA was completely converted to higher molecular weight products at the top of the gel. The ‘‘-” signs indicate the absence of the nucleotide triphosphate, protein, or RNA oligonucleotide.