Non-invasive and high-throughput interrogation of exon-specific isoform expression

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Alternative splicing of mRNA significantly expands the cellular proteome by generating diverse protein isoforms.
• Existing methods for assessing alternative splicing are often labor-intensive, destructive, or lack longitudinal protein-level quantification.
• Understanding exon-specific isoform expression is crucial for cellular function and disease mechanisms.

Purpose of the Study:

• To develop a novel reporter system, EXSISERS, for non-invasive, longitudinal monitoring of exon-specific isoform expression at the protein level.
• To apply EXSISERS for quantifying disease-associated exon inclusion in MAPT and screening RNA-targeting effectors.
• To couple cell survival to specific exon inclusion events and identify regulatory factors.

Main Methods:

• Developed the exon-specific isoform expression reporter system (EXSISERS) utilizing intein-mediated protein splicing for scarless reporter excision.
• Applied EXSISERS to quantify exon 10 inclusion in MAPT using patient-derived induced pluripotent stem cells.
• Screened Cas13-based RNA-targeting effectors for isoform specificity and coupled cell survival to FOXP1 exon 18b inclusion.

Main Results:

• EXSISERS successfully reported translation of exon-containing isoforms non-invasively and with high sensitivity.
• Quantified disease-associated exon 10 inclusion in MAPT and identified specific RNA-targeting effectors.
• Demonstrated coupling of cell survival to FOXP1 exon 18b inclusion and confirmed MBNL1 as a key regulator of its exclusion.

Conclusions:

• EXSISERS provides a non-disruptive, sensitive, and high-resolution method for monitoring exon-specific isoform expression.
• The system facilitates high-throughput screening of therapeutic interventions targeting alternative splicing.
• This technology advances the study of gene isoform regulation in health and disease.