Deciphering BAX and BCL2L12 circRNAs in Acute Myeloid Leukemia Through an Integrated Next-Generation and Nanopore Sequencing Approach

Background: Circular RNAs (circRNAs) constitute an emerging research field, as these RNA molecules play a crucial role in cellular functions and the progression of various human pathologies. Little is known about alternative circularization leading to the formation of distinct circRNAs from the same primary transcript, the role of circRNAs with slightly different back-splice junctions (BSJs) resulting in very similar circRNA sequences—called circRNA isoforms—and the extent to which the same primary transcripts produce alternative circRNAs. In this study, we discovered alternative circRNAs produced by two apoptosis-related genes, BAX and BCL2L12, expressed in established human cell lines originating from myelodysplastic syndrome (MDS) and different types of acute myeloid leukemia (AML). Methods: After total RNA extraction from one MDS cell line and five AML cell lines, first-strand cDNA synthesis, and multiple nested PCRs with distinct sets of divergent primers (10 and 16 primer pairs for BAX and BCL2L12 circRNAs, respectively) annealing in each exon of BAX and BCL2L12 genes, amplicon libraries were prepared and sequenced by both nanopore sequencing and NGS. Detailed bioinformatic analysis was then performed, based on existing bioinformatic tools and our own algorithms. Results: Our approach led to the identification of 72 BAX circRNAs and 52 BCL2L12 circRNAs with distinct expression patterns in MDS and AML cell lines. Most of these circRNAs—either merely exonic or exonic–intronic—were detected for the very first time. Furthermore, several BAX circRNA isoforms were detected in a unique cell line. Moreover, the back-splice sites joined together to form the BSJ of each circRNA were non-canonical, in many cases. The identified circRNAs are predicted to sponge distinct sets of miRNAs, some of which are known to regulate the activity of pivotal pathways. Conclusions: Overall, our findings support the notion that alternative splicing and back-splicing lead to the production of tens of distinct circRNAs from the same human gene.