Molecular misreading: the frequency of dinucleotide deletions in neuronal mRNAs for β-amyloid precursor protein and ubiquitin B

Human neuronal cells contain mutant β-amyloid precursor protein (APP) and ubiquitin B (UBB) mRNAs, in which dinucleotide deletions ('Δ') are generated in/around GAGAG-motifs by an unknown mechanism referred to as 'Molecular Misreading.' The encoded frameshifted (+1) proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD) and in other neurodegenerative and age-related diseases. To measure the concentration of Δ mRNAs, we developed a highly sensitive and specific assay, utilizing peptide nucleic acid-mediated PCR clamping, followed by cloning and colony hybridization with sequence-specific oligonucleotide probes. We found only a few molecules of Δ mRNA/μg of cellular RNA, at levels <10−5 to 10−6 × the concentration of WT mRNA, in RNA extracted from: (i) cultured human neuroblastoma cells grown under a variety of conditions, (ii) the frontal half of brains from wild type and XPA−/− DNA repair-deficient mice, and (iii) post-mortem temporal cortices from humans. Importantly, in RNA from the temporal cortices of AD and Down Syndrome patients that contain βAPP+1 and UBB+1 immunoreactive cells, we found the same low levels of Δ mRNA. We infer that the accumulation of +1 proteins in neurons of these patients is not caused by an increase in the concentration of Δ mRNAs.