FIGURE 3.

Evidence that cytoplasmic sisRNAs are circles (lariats without tails). (A) Plot of average read number per nucleotide for 200 sisRNAs relative to nucleotide position within the intron. Reads abut the 5′ end of the intron, but are absent from a region of ∼30 nt next to the 3′ end. Consensus nucleotides are shown for positions near the ends of the introns. (B) Inverted reads contain sequences that cross the splicing branchpoint and can be mapped when appropriately split (Taggart et al. 2012). (C) Some inverted reads contain sequence errors or nucleotides inserted at the branchpoint (red nucleotides), presumably by the reverse transcriptase used for library production. (D) Cytoplasmic sisRNAs yield an RT–PCR product when amplified with outward-facing primers. These same primers amplify in vitro-transcribed RNA only when the branchpoint is in the interior of the molecule. See text for details of cloning and sequencing of the RT–PCR products.