Is an observed non-co-linear RNA product spliced in trans, in cis or just in vitro?

Global transcriptome investigations often result in the detection of an enormous number of transcripts composed of non-co-linear sequence fragments. Such ‘aberrant’ transcript products may arise from post-transcriptional events or genetic rearrangements, or may otherwise be false positives (sequencing/alignment errors or in vitro artifacts). Moreover, post-transcriptionally non-co-linear (‘PtNcl’) transcripts can arise from trans-splicing or back-splicing in cis (to generate so-called ‘circular RNA’). Here, we collected previously-predicted human non-co-linear RNA candidates, and designed a validation procedure integrating in silico filters with multiple experimental validation steps to examine their authenticity. We showed that >50% of the tested candidates were in vitro artifacts, even though some had been previously validated by RT-PCR. After excluding the possibility of genetic rearrangements, we distinguished between trans-spliced and circular RNAs, and confirmed that these two splicing forms can share the same non-co-linear junction. Importantly, the experimentally-confirmed PtNcl RNA events and their corresponding PtNcl splicing types (i.e. trans-splicing, circular RNA, or both sharing the same junction) were all expressed in rhesus macaque, and some were even expressed in mouse. Our study thus describes an essential procedure for confirming PtNcl transcripts, and provides further insight into the evolutionary role of PtNcl RNA events, opening up this important, but understudied, class of post-transcriptional events for comprehensive characterization.