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

Laura Schulz,Andrei Thomas-Tikhonenko,Katharina E Hayer,Kathi Zarnack,Yoseph Barash,Sarah K Tasian,Manuel Torres-Diz,Julian König,Elena Sotillo,Mukta Asnani,Mariela Cortés-López

doi:10.1186/s13059-021-02411-1

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.

Highlights

Aberrant splicing plays an important role in therapeutic resistance either by generating protein isoforms resistant to treatment or by eliminating target proteins entirely
We previously demonstrated that skipping of exon 2 of CD19 pre-mRNA generates a protein variant inherently resistant to killing by CART-19 and mis-localized in the endoplasmic reticulum [2, 3]
Close examination of the CD19 exon 2 sequence revealed that the putative exitron could be folding into a stable hairpin flanked by two 8-nt direct repeats (Fig. 1b), hinting at possible reverse transcription (RT) or PCR slippage at the base of the hairpin and ensuing product truncation

Summary

Introduction

Aberrant splicing plays an important role in therapeutic resistance either by generating protein isoforms resistant to treatment or by eliminating target proteins entirely. A prime example of this phenomenon is B cell acute lymphoblastic leukemia (B-ALL) acquiring resistance to chimeric antigen receptor-armed autologous T cells (CART-19), which are engineered to target the CD19 surface antigen of B cells [1]. We previously demonstrated that skipping of exon 2 of CD19 pre-mRNA generates a protein variant inherently resistant to killing by CART-19 and mis-localized in the endoplasmic reticulum [2, 3]. We and others have shown that retention of the CD19 intron 2 containing a premature termination codon contributes to CART-19 resistance as well [4, 5]. Several publications reported that apparent removal of a cryptic intron fully embedded within CD19 exon 2 generates a novel isoform in healthy individuals and B-ALL patients (termed Δex2part) [2, 6,7,8]. One study further suggested that this event could mediate resistance to blinatumomab, a CD19-CD3-bispecific T

Methods

Results

Conclusion