Long-Read Sequencing Reveals Cell- and State-Specific Alternative Splicing in 293T and A549 Cell Transcriptomes

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Alternative splicing (AS) generates transcriptomic diversity, influencing cellular identity.
• Cell-type-specific AS, especially in response to receptor signaling, is not fully understood in common cell lines like 293T and A549.
• Understanding AS dynamics is key to interpreting cellular function and disease states.

Purpose of the Study:

• To characterize cell-type-specific alternative splicing dynamics in 293T and A549 cells under basal and stimulated conditions.
• To investigate the impact of G protein-coupled receptor (GPCR) overexpression on transcriptomic complexity and splicing patterns.
• To evaluate the utility of integrating long-read sequencing with bioinformatic tools for comprehensive AS analysis.

Main Methods:

• Integrated Oxford Nanopore long-read sequencing with BGI short-read sequencing.
• Profiled transcriptomes of 293T and A549 cells under basal and GPCR-overexpressing conditions (ADORA3 in 293T, P2RY12 in A549).
• Performed full-length isoform analysis using FLAIR and SQANTI3.

Main Results:

• Discovered extensive transcriptomic complexity, with 18.02% novel isoforms in 293T and 19.52% in A549 cells.
• Observed a stable transcriptome in 293T cells, enriched in splicing-related pathways.
• Identified broader transcriptional remodeling in A549 cells associated with tumorigenic processes.

Conclusions:

• 293T cells are a suitable model for studying splicing regulation due to their stable transcriptome.
• A549 cells provide a relevant model for exploring tumor-associated transcriptome dynamics.
• Integrating long-read sequencing with FLAIR/SQANTI3 is valuable for dissecting cell-state-specific AS regulation.