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      Evidence That an Endothelial Cytosolic Protein Binds to the 3′-Untranslated Region of Endothelial Nitric Oxide Synthase mRNA

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          Changes in the endothelial nitric oxide synthase (eNOS) expression could be involved in the endothelium-dependent vasorelaxing dysfunction associated with cardiovascular diseases. We have recently demonstrated the existence of endothelial cytosolic proteins that bind to the 3′-untranslated region (3′-UTR) of eNOS mRNA and could be involved in eNOS mRNA stabilization. In the present work, we have characterized the cytosolic proteins that bind to 3′-UTR eNOS mRNA. An endothelial cytosolic protein (MW 60-kD) specifically bound to 3′-UTR eNOS mRNA as determined by a cross-linking assay followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The endothelial cytosolic protein recognized a cytidine (C)-rich region within 3′-UTR eNOS mRNA. Furthermore, tumor necrosis factor-α (TNF-α) increased the level of the 60-kD endothelial cytosolic protein. In addition, TNF-α reduced eNOS mRNA levels and this was prevented by coincubation with cycloheximide. Cycloheximide also prevented the binding activity of the endothelial cytosolic protein to 3′-UTR eNOS mRNA. In summary, these data suggest that a 60-kD endothelial cytosolic protein binds to 3′-UTR eNOS mRNA. TNF-α increased the 60-kD protein levels. Cycloheximide prevented the binding activity of the cytosolic protein to 3′-UTR eNOS mRNA related to TNF-α; this effect was associated with greater eNOS mRNA levels. Further specific studies are needed to determine the involvement of this 60-kD endothelial cytosolic protein in the regulation of eNOS mRNA stabilization and in the endothelial dysfunction associated with cardiovascular diseases.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Nucleolin and heterogeneous nuclear ribonucleoprotein C proteins specifically interact with the 3'-untranslated region of amyloid protein precursor mRNA.

            The central nervous system deposition by neurons and glia of beta A4 amyloid protein is an important contributing factor to the development of Alzheimer's disease. Amyloidogenic cells overexpress amyloid precursor protein (APP) mRNAs suggesting a transcriptional or post-transcriptional defect may contribute to this process. We have previously shown that APP mRNAs display regulated stability which is dependent on a 29-base element within the 3'-untranslated region (UTR). This domain specifically interacted with several cytoplasmic RNA-binding proteins. We have purified these APP RNA-binding proteins from a human T-cell leukemia and demonstrate that five cytoplasmic proteins of 70, 48, 47, 39, and 38 kDa form the previously observed APP RNA protein complexes. Amino acid sequence analyses showed that the 70-, 48-, and 47-kDa proteins were fragments of nucleolin and that the 39- and 38-kDa proteins were heterogeneous nuclear ribonucleoprotein (hnRNP) C protein. Northwestern and Western blot analyses of purified material further confirmed these data. Nucleolin protein is known to shuttle between the nucleus and cytoplasm but hnRNP C has not been reported within the cytoplasm. This report of sequence specific, mRNA binding by nucleolin and hnRNP C suggests that these proteins participate in the post-transcriptional regulation of APP mRNA through 3'-UTR, site-specific interactions.

              Author and article information

              J Vasc Res
              Journal of Vascular Research
              S. Karger AG
              June 1999
              18 June 1999
              : 36
              : 3
              : 201-208
              aNephrology, Hypertension and Cardiovascular Research Laboratory, Fundación Jiménez Díaz, Madrid and bDepartment of Biochemistry and Cellular and Molecular Biology, Faculty of Veterinary Medicine, University of Zaragoza, Zaragoza, Spain
              25643 J Vasc Res 1999;36:201–208
              © 1999 S. Karger AG, Basel

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              Page count
              Figures: 7, References: 25, Pages: 8
              Research Paper


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