Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts

• Published in: Genome Biology Vol. 22; no. 1; p. 190
• Main Authors: Schulz, Laura, Torres-Diz, Manuel, Cortés-López, Mariela, Hayer, Katharina E, Asnani, Mukta, Tasian, Sarah K, Barash, Yoseph, Sotillo, Elena, Zarnack, Kathi, König, Julian, Thomas-Tikhonenko, Andrei
• Format: Journal Article
• Language: English
• Published: England BioMed Central 28-06-2021; BMC
• Subjects: Alternative Splicing; Antibodies, Bispecific - pharmacology; Antigens; Antineoplastic Agents, Immunological - pharmacology; B-Lymphocytes - drug effects; B-Lymphocytes - immunology; B-Lymphocytes - pathology; Base Pairing; Base Sequence; Cancer; CD19 antigen; Cell Line, Tumor; Datasets as Topic; Exitrons; Exons; High-Throughput Nucleotide Sequencing; Humans; Hypotheses; Immunotherapy; Immunotherapy - methods; Introns; Long-read sequencing; Lymphatic leukemia; Models, Biological; mRNA isoforms; Nucleic Acid Conformation; Oxford Nanopore Technologies; Precursor Cell Lymphoblastic Leukemia-Lymphoma - drug therapy; Precursor Cell Lymphoblastic Leukemia-Lymphoma - genetics; Precursor Cell Lymphoblastic Leukemia-Lymphoma - immunology; Precursor Cell Lymphoblastic Leukemia-Lymphoma - pathology; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - immunology; Proteins; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - immunology; Reverse Transcription; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - immunology; Short Report; Alternative splicing; Exitrons; mRNA isoforms; Blinatumomab; CD19; Immunotherapy; Long-read sequencing; Reverse transcription; Oxford Nanopore Technologies
• ISSN: 1474-760X; 1474-7596; 1474-760X
• DOI: 10.1186/s13059-021-02411-1

Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, 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.