Investigating lariat intron RNA candidates for therapeutic RNA aptamer targeting specific dsRNA sensors

Abstract

In eukaryotic organisms, introns are universally present and constitute a distinctive feature. Following RNA transcription, pre-mRNAs undergo an intron-splicing process, leading to the removal of introns in the form of a lariat. These excised introns are targeted by exonucleases and degraded following debranching, leading to their classification as gene expression byproducts. We found that the deficiency of intron debranching enzyme DBR1 can induce the accumulation of lariat-form introns in cells. The accumulated lariat-form introns then form double-stranded RNA (dsRNA) structure based on Alu sequences that they are hosting, which binds with protein kinase R (PKR) and induces cell apoptosis, which can be a potential cancer therapeutics. Furthermore, we found that dsRNA structure formed by lariat-form introns preferentially activated PKR over other dsRNA sensors such as melanoma-differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I), which results in weak induction of cellular interferon responses. By optimizing in vitro binding experiments with FLAG-tagged PKR and MDA5 and shape-retained (lariat) or debranched (linear) intronic RNAs, we analyze specific RNA intron species that bind to each dsRNA sensor by RNA sequencing.