Abstract
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.
Keywords:
Alternative splicing; Blinatumomab; CD19; Exitrons; Immunotherapy; Long-read sequencing; Oxford Nanopore Technologies; Reverse transcription; mRNA isoforms.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alternative Splicing*
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Antibodies, Bispecific / pharmacology
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Antineoplastic Agents, Immunological / pharmacology
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Artifacts*
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B-Lymphocytes / drug effects
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B-Lymphocytes / immunology
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B-Lymphocytes / pathology
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Base Pairing
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Base Sequence
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Cell Line, Tumor
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Datasets as Topic
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Exons
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High-Throughput Nucleotide Sequencing
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Humans
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Immunotherapy / methods
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Introns
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Models, Biological
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Nucleic Acid Conformation
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / drug therapy
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / genetics
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / immunology
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / pathology
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Protein Isoforms / chemistry
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Protein Isoforms / genetics
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Protein Isoforms / immunology
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RNA, Messenger / chemistry
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RNA, Messenger / genetics*
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RNA, Messenger / immunology
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Receptors, Antigen, T-Cell / genetics*
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Receptors, Antigen, T-Cell / immunology
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Reverse Transcription*
Substances
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Antibodies, Bispecific
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Antineoplastic Agents, Immunological
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CD19-specific chimeric antigen receptor
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Protein Isoforms
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RNA, Messenger
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Receptors, Antigen, T-Cell
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blinatumomab