Reference - Detail
|Author||Zimowska G, Shi J, Munguba G, Jackson MR, Alpatov R, Simmons MN, Shi Y, Sugrue SP.|
|Title||Pinin/DRS/memA interacts with SRp75, SRm300 and SRrp130 in corneal epithelial cells.|
|Journal||Invest. Ophthalmol. Vis. Sci.|
PURPOSE:Pinin (Pnn/DRS/memA) is a cell-adhesion-related and nuclear protein that has been identified as central in the establishment and maintenance of corneal epithelial cell-cell adhesion. To begin the elucidation of the role of Pnn within the nucleus of corneal epithelial cells, this study was undertaken to identify the proteins that bind to Pnn.
METHODS:Yeast two-hybrid analyses were performed. A human cDNA library in the pGAD-10 vector and C-terminal region of human Pnn (465-717) in a pAS2-1 vector were cotransformed into the PJ69-4A yeast strain, containing the lacZ, HIS3, and ADE2 reporter genes. To dissect domains of Pnn responsible for mediating the interaction with the identified proteins, PNN fragments were ligated with the DNA-binding domain of the pAS2-1 vector. Human corneal epithelial cells (HCE-T, RCB1384) and HEK-293 cells were cotransfected with mammalian expression vectors containing Pnn with identified interacting partners and subsequently immunostained and immunoblotted to determine expressed and endogenous proteins.
RESULTS:Pnn colocalized and copurified with serine-arginine (SR) proteins. Three SR-rich proteins were identified that interact with the C-terminus of Pnn: SRp75 and SRm300, known components of spliceosome machinery, and a novel 130-kDa nuclear protein, SRrp130. All of these proteins colocalized and coimmunoprecipitated with one another and exhibited speckled nuclear distribution that aligned with components of the pre-mRNA splicing machinery. The cDNA for SRrp130 encoded a protein of 805 amino acid residues and contained multiple arginine-serine (RS) repeats but had no RNA recognition motif. Analysis of the Pnn motifs using two-hybrid system assays demonstrated that the polyserine/RS motif within Pnn plays a central but not exclusive role in mediating molecular interactions with identified SR-rich proteins.
CONCLUSIONS:The results suggest that Pnn and SR-rich proteins may be part of a multiprotein complex within the nucleus and may be involved in pre-mRNA processing.
|MeSH||Amino Acid Sequence Carrier Proteins / genetics Carrier Proteins / metabolism* Cell Adhesion Molecules / genetics Cell Adhesion Molecules / metabolism* Cloning, Molecular Epithelium, Corneal / metabolism* Gene Library Genetic Vectors HeLa Cells Humans Immunoblotting Molecular Sequence Data Nuclear Proteins / genetics Nuclear Proteins / metabolism* Plasmids Precipitin Tests Protein Binding RNA Splicing / genetics RNA-Binding Proteins / genetics RNA-Binding Proteins / metabolism Saccharomyces cerevisiae / genetics Serine-Arginine Splicing Factors Transfection Two-Hybrid System Techniques|
|Human and Animal Cells||HCE-T (RCB1384)|