Exploring U6atac Inhibition and Minor Intron Splicing Dynamics for Therapeutic Strategies

Intron splicing is crucial for gene expression in eukaryotes, and minor introns (mi-INTs) represent a distinct class reliant on U6atac snRNP. Despite their rarity, mi-INTs are highly conserved across species and are enriched in genes involved in key cellular processes, including those implicated in cancer. We hypothesized that deregulated splicing of mi-INTs contributes to the aggressiveness and therapeutic resistance seen in breast cancer. Using antisense oligonucleotides, we knocked down U6atac in two subtypes of breast cancer cells, measuring relative gene expression via qPCR and conducting cell viability assays. Complete U6atac inactivation led to rapid and severe cell toxicity, revealing distinct splicing trends in 23 genes. Surprisingly, mild U6atac inhibition also decreased cell viability significantly without affecting all mi-INTs splicing, suggesting a set of highly sensitive genes essential for breast cancer cell survival, potentially viable targets for low-dose inhibition strategies. This study had three goals aimed at understanding breast cancer cell responses to U6atac inhibition and elucidating underlying mechanisms. First. We assessed viability differences in breast cancer cells following moderate U6atac inhibition and investigated cell cycle arrest and apoptosis. Then, we aimed to identify and characterize minor intron- containing genes responsive to U6atac inhibition through RNA-Seq screening and computational analysis in order to discern motif differences between breast cancer subtypes. Finally, we explored the lack of transcript accumulation in some mi-INTs by inhibiting the exosomal and NMD pathways, seeking to unravel interactions and splicing behaviors. This research provided insights into molecular mechanisms underlying breast cancer cell responses to U6atac inhibition, potentially uncovering therapeutic targets and enhancing understanding of breast cancer pathogenesis.