Molecular misreading: the frequency of dinucleotide deletions in neuronal mRNAs for beta-amyloid precursor protein and ubiquitin B.
Human neuronal cells contain mutant beta-amyloid precursor protein (APP) and ubiquitin B (UBB) mRNAs, in which dinucleotide deletions ('Delta') 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 Delta 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 Delta mRNA/microg of cellular RNA, at levels <10(-5) to 10(-6) x 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 betaAPP+1 and UBB+1 immunoreactive cells, we found the same low levels of Delta mRNA. We infer that the accumulation of +1 proteins in neurons of these patients is not caused by an increase in the concentration of Delta mRNAs.
Gerez
Lisya
L
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands.
de Haan
Annett
A
Hol
Elly M
EM
Fischer
David F
DF
van Leeuwen
Fred W
FW
van Steeg
Harry
H
Benne
Rob
R
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004314:Down Syndrome / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D008967:Molecular Biology / Q000379:methods; D009474:Neurons / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods; D011180:Postmortem Changes; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D051760:Xeroderma Pigmentosum Group A Protein
D016564:Amyloid beta-Protein Precursor; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D012333:RNA, Messenger; C479419:UBB protein, human; D025801:Ubiquitin; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C497315:Xpa protein, mouse