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The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1 specifically interact with an exon 3-derived
R J Kelm1, P K Elder, M J Getz
1Department of Biochemistry, Molecular Biology Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA. kelm.robert@mayo.edu
Abstract:
Amino acids 44-53 of mouse vascular smooth muscle alpha-actin are encoded by a region of exon 3 that bears structural similarity to an essential MCAT enhancer element in the 5' promoter of the gene. The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, interact with each other and with opposite strands of the enhancer to repress transcription in fibroblasts (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436; Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). In this study, we employed both recombinant and fibroblast-derived proteins to demonstrate that all three proteins specifically interact with the mRNA counterpart of the exon 3 sequence in cell-free binding assays. When placed in the 5'-untranslated region of a reporter mRNA, the exon 3-derived sequence suppressed mRNA translation in transfected fibroblasts. Translational efficiency was restored by mutations that impaired mRNA binding of Puralpha, Purbeta, and MSY1, implying that these proteins can also participate in messenger ribonucleoprotein formation in living cells. Additionally, primary structure determinants required for interaction of Purbeta with single-stranded DNA, mRNA, and protein ligands were mapped by deletion mutagenesis. These experiments reveal highly specific protein-mRNA interactions that are potentially important in regulating expression of the vascular smooth muscle alpha-actin gene in fibroblasts.
Insights
Single-stranded DNA-binding proteins Puralpha, Purbeta, and MSY1 bind to the alpha-actin gene
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein-DNA/RNA Interactions
Background:
- Vascular smooth muscle alpha-actin gene expression is crucial for cell function.
- Specific DNA-binding proteins (Puralpha, Purbeta, MSY1) are known to interact with an enhancer element in the gene's promoter.
- These proteins were previously shown to repress gene transcription.
Purpose of the Study:
- To investigate the interaction of Puralpha, Purbeta, and MSY1 with the mRNA of the vascular smooth muscle alpha-actin gene.
- To determine if these proteins regulate gene expression at the post-transcriptional level.
- To map the protein-binding determinants within the Purbeta protein.
Main Methods:
- Cell-free binding assays using recombinant and fibroblast-derived proteins.
- Reporter mRNA assays in transfected fibroblasts.
- Deletion mutagenesis to map protein interaction domains.
Main Results:
- Puralpha, Purbeta, and MSY1 specifically bind to the mRNA counterpart of the exon 3 sequence.
- The exon 3 sequence, when in the 5'-untranslated region, suppresses mRNA translation.
- Mutations disrupting protein binding restore translational efficiency, indicating a role in messenger ribonucleoprotein formation.
- Key interaction sites on Purbeta for DNA, mRNA, and protein ligands were identified.
Conclusions:
- Puralpha, Purbeta, and MSY1 engage in specific protein-mRNA interactions.
- These interactions can regulate the translation of vascular smooth muscle alpha-actin mRNA.
- The findings suggest a novel mechanism for gene expression control involving post-transcriptional regulation by these proteins.