How we used MPAT-Seq to study pathogenic mutations in the PDE12 gene

Introduction

Mitochondrial function is pivotal for energy metabolism, with defects often resulting in severe multisystem disorders. Phosphodiesterase 12 (PDE12), a poly(A)-specific exoribonuclease, is integral to mitochondrial RNA quality control, particularly in removing non-templated poly(A) tails from mitochondrial non-coding RNAs (mt-tRNAs and mt-rRNAs). Using Mitochondrial Poly(A)-Tail RNA Sequencing (MPAT-Seq), we identified the pathogenic mechanisms linked to PDE12 mutations in affected patients, revealing its critical regulatory role in mitochondrial RNA stability and OXPHOS function.

The Need for MPAT-Seq in Mitochondrial RNA Studies

Pathogenic missense variants in PDE12 have been implicated in spurious polyadenylation of mitochondrial RNAs, a defect that destabilises mitochondrial RNA processing. Traditional methods were insufficient to resolve these RNA extensions accurately, necessitating MPAT-Seq, a high-resolution sequencing technique designed to detect and quantify aberrant poly(A) tail additions in mitochondrial RNA.

Methodology: Applying MPAT-Seq to PDE12 Research

1. Sample Preparation

   • RNA was extracted from patient-derived fibroblasts, liver samples, and PDE12-knockout HEK293T cells.

   • RNA was ligated with a primer to capture polyadenylation, followed by reverse transcription and PCR amplification.

2. High-Throughput Sequencing

   • RNA libraries were sequenced using Illumina platforms, ensuring precise mapping of mitochondrial RNA 3’ ends.

3. Bioinformatics Analysis

   • Custom computational pipelines identified spurious polyadenylation events, enabling a comparison between patient and control samples.

You can find examples here or read the full manuscripts: Pearce et al., 2017 and Van Haute et al., 2024. Or check our video here