Structural characterization and expression analysis of novel MAPK1 transcript variants with the development of a multiplexed targeted nanopore sequencing approach

Mitogen-activated protein kinases (MAPKs) represent a protein family firmly involved in many signaling cascades, regulating a vast spectrum of stimulated cellular processes. Studies have shown that alternatively spliced isoforms of MAPKs play a crucial role in determining the desired cell fate in response to specific stimulations. Although the implication of most MAPKs transcript variants in the MAPK signaling cascades has been clarified, the transcriptional profile of a pivotal member, MAPK1, has not been investigated for the existence of additional isoforms. In the current study we developed and implemented targeted long-read and short-read sequencing approaches to identify novel MAPK1 splice variants. The combination of nanopore sequencing and NGS enabled the implementation of a long-read polishing pipeline using error-rate correction algorithms, which empowered the high accuracy of the results and increased the sequencing efficiency. The utilized multiplexing option in the nanopore sequencing approach allowed not only the identification of novel MAPK1 mRNAs, but also elucidated their expression profile in multiple human malignancies and non-cancerous cell lines. Our study highlights for the first time the existence of ten previously undescribed MAPK1 mRNAs (MAPK1 v.3 - v.12) and evaluates their relative expression levels in comparison to the main MAPK1 v.1. The optimization and employment of qPCR assays revealed that MAPK1 v.3 - v.12 can be quantified in a wide spectrum of human cell lines with notable specificity. Finally, our findings suggest that the novel protein-coding mRNAs are highly expected to participate in the regulation of MAPK pathways, demonstrating differential localizations and functionalities.