Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.
Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
Genome Biol. 2021 Jun 28;22(1):190. 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.
在淋巴母细胞性白血病中，针对 CD19 的免疫疗法产生耐药性的原因除其他因素外，还包括几个异常的 CD19 前体 mRNA 剪接事件，包括最近报道的 CD19 外显子 2 内嵌入的隐秘内含子的切除。虽然已知“出口子”存在于数百个人类转录本中，但我们使用报告基因检测和直接长读 RNA 测序（dRNA-seq）发现，CD19 出口子是逆转录的人为产物。通过将我们的分析扩展到公开可用的数据集，我们鉴定了数十个有问题的出口子，称为“假出口子”，它们仅出现在 cDNA-seq 中，而从未出现在 dRNA-seq 中。我们的结果强调了 dRNA-seq 对于转录本异构体验证的重要性。
