PRJNA725229
Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts
Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other things, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While exitrons are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons (dubbed falsitrons) that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.