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P2‐153: SIRT1 is decreased in Alzheimer's disease and correlates with tau pathology
Author(s) -
Julien Carl,
Tremblay Cyntia,
Phivilay Alix,
Rancourt Caroline,
Calon Frédéric
Publication year - 2008
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2008.05.1227
Subject(s) - hippocampus , cortex (anatomy) , posterior parietal cortex , alzheimer's disease , endocrinology , medicine , biology , cerebellum , in situ hybridization , sirtuin 1 , cerebral cortex , temporal cortex , tau protein , amyloid precursor protein , messenger rna , neuroscience , disease , downregulation and upregulation , biochemistry , gene
element in the 3’ untranslated region (3’UTR) known as the axonal targeting element (ATE). The ATE contains a binding site for HuD, a member of the embryonic lethal abnormal vision like family. HuD stabilises tau mRNA by binding a U-rich regulatory region controlling mRNA turnover. The full complement of trans-acting factors binding to the ATE responsible for the specific localisation of tau mRNA to axons is still to be identified. The objective of this study is to analyse the mechanisms regulating tau mRNA subcellular localisation by first identifying ATE-binding proteins using proteomic analysis. Methods: Trans-acting factors which bind the 3’UTR of tau mRNA were isolated using a novel RNA affinity purification procedure. We have designed the pOC vector, in a pBS backbone, containing a 19 bp tagging sequence adjacent to the BamHI site into which a DNA sequence encoding the ATE is subcloned. In-vitro transcription from the T3 promoter produces RNA tagged at the 3’ end. Tagged RNA is annealed to a biotinylated oligoribonucleotide corresponding to the tag sequence in the antisense orientation. This oligoribonucleotide is synthesised with modified 2’-O-methyl bases increasing the stability of the RNA-RNA duplex and protecting it from RNase degradation. An immobilised tagged RNA matrix is prepared by binding the biotinylated RNA to magnetic streptavidin beads. Results: This novel protocol was validated by the specific isolation of HuD from cell extracts overexpressing recombinant HuD. Large scale studies were performed to isolate endogenous RNA-binding proteins from adult rat brain. We observe specific binding of endogenous trans-acting factors binding to the ATE of tau mRNA. Moreover, these proteins are isolated in amounts compatible with their identification by mass spectrometry; allowing global identification of tau ATE-binding proteins. Conclusions: Understanding the mechanisms by which RNA-binding proteins regulate tau mRNA processing will lead to increased understanding of the pathology of tauopathies and the development of novel therapeutic strategies.