Bioinformatics-driven refinement of the commonly used TPI nonsense-mediated decay reporter system

Area of Science:

• Molecular Biology
• RNA Biology
• Genetics

Background:

• The nonsense-mediated decay (NMD) pathway is a crucial cellular mechanism for degrading aberrant mRNAs, acting as a transcript quality control system.
• NMD pathway research heavily relies on reporter systems, typically multiexon constructs with wild-type and premature termination codon variants.
• Previous studies using the triose phosphate isomerase (TPI) reporter revealed unexpected cryptic splice site usage, potentially compromising reporter reliability.

Purpose of the Study:

• To investigate the impact of cryptic splice site usage on NMD reporter accuracy.
• To develop strategies for enhancing the reliability and specificity of NMD reporter systems.
• To provide a generalizable method for improving minigene construction for studying cellular transcripts.

Main Methods:

• Utilized a seven-exon TPI reporter system to identify non-constitutive splice site usage.
• Modified the TPI reporter by restoring intron length and performing bioinformatic adjustments to splice regulatory elements (SREs) and splice site strength.
• Evaluated the impact of these modifications on reporter robustness and specificity for NMD sensitivity.

Main Results:

• Observed the unexpected use of cryptic splice sites in the standard TPI NMD reporter.
• Demonstrated that restoring intron length and optimizing SREs/splice site strength significantly improved reporter performance.
• The modified TPI reporter exhibited enhanced robustness and specificity for evaluating NMD sensitivity.

Conclusions:

• Cryptic splice site usage can undermine the reliability of NMD reporter systems.
• Minigene construction can be optimized to ensure constitutive splice site recognition, leading to more accurate NMD assessment.
• The presented modifications offer a generalizable approach for improving multiexon transcript minigenes for NMD studies.