Integrated Exon and Intron Splicing Analysis Identifies Novel GAS5 and PVT1 Regulatory Junctions in Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EAC) exhibits widespread transcriptional dysregulation, but the contribution of noncoding RNA splicing remains poorly characterized. Publicly available RNAseq datasets comprising primary EAC epithelial cell lines and normal esophageal mucosa were analyzed using a fully reproducible pipeline integrating STAR alignment, Cuffdiff expression modeling, and joint exon-centric (rMATS) and intron centric (MntJULiP) splicing detection. After splicing burden and sample level residualization, the bias corrected expression ranking was dominated by extracellular matrix and stress response genes. A genome wide splicing frequency map was constructed for noncoding genes, revealing several lncRNAs with high event density. A stringent concordance filter requiring simultaneous rMATS significance (FDR ≤ 0.05, |ΔPSI| ≥ 0.20), MntJULiP significance (q ≤ 0.05), and genomic co localization identified GAS5 as the top noncoding splicing hotspot. Coordinate level analysis revealed a compact RI/MXE/A5SS cluster at chr1:173.865 Mb, annotated as an exact intron in GENCODE v40 but showing negligible junction support in GTEx normal esophageal tissues. The same configuration was not explicitly represented in TCGA PanCancer Atlas splicing catalogs, indicating that this structurally annotated event has remained under reported despite biological plausibility. In addition, a previously unreported PVT1 SE/MXE splice junction (chr8:128,807,020 128,807,311) present exclusively in EAC tumor RNA and absent in GTEx and TCGA PanCancer Atlas junction catalogs was also discovered.This work provides a fully documented and reproducible framework for discovering noncoding splicing alterations in EAC, reveals new coordinate resolved targets for mechanistic and therapeutic investigation and nominates a GAS5 intron exon configuration and a previously unreported PVT1 SE/MXE splice junction as a potential regulator of EAC biology highlighting deficiencies in the TCGA database.