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

Area of Science:

• Molecular Biology
• Genomics
• Immunotherapy

Background:

• Resistance to CD19-directed immunotherapies in lymphoblastic leukemia is a significant clinical challenge.
• Aberrant CD19 pre-mRNA splicing, including cryptic intron excision, has been implicated in treatment failure.
• The existence and functional relevance of specific RNA splicing events require rigorous validation.

Purpose of the Study:

• To investigate the nature of a reported CD19 "exitron" and its role in lymphoblastic leukemia.
• To determine the accuracy of RNA sequencing methods in identifying true transcript isoforms.
• To establish reliable methods for transcript isoform validation in complex biological samples.

Main Methods:

• Reporter assays were employed to functionally assess RNA splicing events.
• Direct long-read RNA sequencing (dRNA-seq) was utilized for high-resolution transcript analysis.
• Publicly available cDNA sequencing datasets were re-analyzed to identify potential artifacts.

Main Results:

• The CD19 "exitron" was identified as an artifact arising during reverse transcription, not a genuine spliced transcript.
• Analysis revealed numerous "falsitrons" (artifactual exitrons) present in cDNA sequencing data but absent in dRNA-seq.
• dRNA-seq proved essential for distinguishing true splice variants from reverse transcription artifacts.

Conclusions:

• The previously reported CD19 exitron is an artifact, impacting our understanding of immunotherapy resistance mechanisms.
• The study underscores the critical importance of dRNA-seq for accurate transcript isoform identification and validation.
• This highlights potential pitfalls in interpreting cDNA-based sequencing data for RNA splicing studies.