Exon skipping as a potential diagnostic biomarker in colorectal cancer: an integrated epigenomic-transcriptomic...

Colorectal cancer (CRC) is the third most common cancer globally. Alternative splicing contributes significantly to CRC tumorigenesis through aberrant transcript generation. However, the regulatory influence of RNA modifications on splicing remains poorly understood, largely due to technical difficulty. Nanopore direct RNA sequencing addresses this by enabling simultaneous detection of RNA modifications and Alternative splicing events (ASEs).

We conducted Nanopore direct RNA sequencing on paired tumor and normal tissues from surgical resections at Beijing Hospital. Differential putative RNA modification sites and ASEs linked to CRC were systematically identified. To validate the key findings, we utilized a large patient cohort from The Cancer Genome Atlas (TCGA) and predicted 3D protein structures with AlphaFold3. The predicted structures were then compared using TM-align. Regulatory relationships between RNA modifications and splicing were explored through predictive modeling of potential cis-regulatory pairs. The splicing events were also validated.

The MYH11-201 transcript of the MYH11 gene contains an additional exon (ENSE00001632812) compared to the MYH11-203 isoform. Both bioinformatic analysis and experimental validation confirmed frequent loss of this exon in tumor tissues. This finding was further validated in the TCGA cohort, demonstrating a significant preference for exon skipping in tumor tissues. These results suggest that the skipping of ENSE00001632812 is a promising candidate biomarker associated with CRC pathogenesis. Notably, this exon's PF00063 domain interacts with multiple tumor suppressor genes and oncogenes domains, suggesting its functional importance. The structures revealed pronounced rotational divergence within a putative C-terminal transmembrane domain-like region. Furthermore, we utilized Nanopore sequencing to explore the potential interplay between alternative splicing and RNA modifications. We implemented an integrated analytical workflow (available at https://github.com/lelelililele/Nanopore-ASEs-and-RNA-modification ) combining modification calling and splicing analysis tools to investigate RNA modification-related enzymes and splicing-related proteins in CRC.

This pilot study utilizes Nanopore direct RNA sequencing to characterize exon skipping events and RNA modifications in CRC. We identified the skipping of MYH11 exon ENSE00001632812 as a potential candidate for future diagnostic investigation. By integrating modification and splicing data, we highlighted putative regulatory pairs that warrant further functional exploration. While our findings offer new insights into CRC molecular mechanisms, extensive validation in independent large-scale cohorts and functional assays is essential to confirm the diagnostic utility and mechanistic roles of these targets.