PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Next-generation sequencing (NGS) identifies numerous genetic variants impacting microRNA (miRNA)-mRNA interactions.
• Functional validation of these variants, particularly those in miRNA binding sites (mirSNPs), is crucial but challenging.
• Existing methods are often low-throughput, limiting comprehensive analysis.

Purpose of the Study:

• To develop and validate a novel high-throughput bioassay, PASSPORT-seq, for functional testing of mirSNPs.
• To assess the cell-type specificity of mirSNP function.
• To investigate the functional impact of mirSNPs in pharmacogenes.

Main Methods:

• Development of PASSPORT-seq, a high-throughput assay for parallel functional testing of hundreds of mirSNPs.
• Application of PASSPORT-seq to test 100 mirSNPs across HEK293, HepG2, and HeLa cell lines.
• Validation of selected results using traditional individual luciferase assays.
• Functional testing of 111 variants in the 3' UTR of 17 pharmacogenes.

Main Results:

• PASSPORT-seq demonstrated high reproducibility across technical and biological replicates.
• Fifty-five out of 100 tested mirSNPs were functional in at least one cell line (FDR ≤ 0.05).
• Significant cell-type specificity was observed, with only 4 variants functional in all three tested cell lines (HEK293, HepG2, HeLa).
• Thirty-three of 111 variants in pharmacogenes showed functional effects in at least one cell line.

Conclusions:

• PASSPORT-seq is a robust and reproducible high-throughput method for functional mirSNP analysis.
• MirSNP function is frequently cell-type specific, underscoring the need for multi-cell line testing.
• The assay is valuable for identifying functional variants in pharmacogenes and understanding miRNA regulation.