Analysis of miRNA Modifications.

After transcription, a large number of cellular RNAs employ modifications to increase their diversity and functional potential. Modifications can occur on the base, ribose, or both, and are important steps in the maturation of many RNAs. Our lab recently showed that plant microRNAs (miRNAs) possess a 2'-O-methyl group on the ribose of the 3' terminal nucleotide, and that this methyl group is added after miRNA/miRNA* formation. One function of this modification is to protect miRNAs from 3' terminal uridylation by an unknown enzymatic activity. It is possible that uridylation of miRNAs triggers their degradation. Here we describe a protocol to purify a specific miRNA in order to determine its molecular mass so that the presence of a modification can be inferred, an in vivo method to detect 3' terminal modification of miRNAs, and an (alpha-32P) dATP incorporation assay to study 3' terminal uridylation of miRNAs.