Figure 2. The mating-specific 27 nt RNAs in Oxytricha trifallax are not modified at their 3′ ends.

RNAs were tested with the beta elimination assay in order to determine if there is a modification at the 3′ end. RNAs tested are ³²P 5′ end labeled total Oxytricha trifallax RNA (lanes 2–11) or 5′ end labeled synthetic positive control RNAs containing the sequence of the C. elegans lin-4 (21 nt) or mir-90 (23 nt) microRNAs which were subsequently mixed with 1.5 µg of unlabeled Oxytricha total RNA prior to beta elimination (lanes 13–16). Control untreated samples (-) or samples subjected to the beta elimination reaction (+) are indicated. RNAs were isolated from vegetative ALXC2 (lanes 2 and 3) or from a mating between ALXC2 and ALXC9 that were harvested at the indicated timepoint after mixing (lanes 4–11). Beta elimination will remove the terminal ribose if both a free 2′ OH and 3′ OH are present, and will leave a 3′ cyclic phosphate. This loss of a nucleotide and the presence of the extra phosphate group will result in the treated RNAs running almost two bases faster than their untreated counterparts.