RESUMO
Technological advantages in sequencing and proteomics have revealed the remarkable diversity of alternative protein isoforms. Typically, the localization and functions of these isoforms are unknown and cannot be predicted. Also the localization signals leading to particular subnuclear compartments have not been identified and thus, predicting alternative functions due to alternative subnuclear localization is limited only to very few subnuclear compartments. Knowledge of the localization and function of alternative protein isoforms allows for a greater understanding of cellular complexity. In this article, we characterize a short and well-defined signal targeting the bovine papillomavirus type 1 E8/E2 protein to the nuclear matrix. The targeting signal comprises the peptide coded by E8 ORF, which is spliced together with part of the E2 ORF to generate the E8/E2 mRNA. Localization to the nuclear matrix correlates well with the transcription repression activities of E8/E2; a single point mutation directs the E8/E2 protein into the nucleoplasm, and transcription repression activity is lost. Our data prove that adding as few as Ë10 amino acids by alternative transcription/alternative splicing drastically alters the function and subnuclear localization of proteins. To our knowledge, E8 is the shortest known nuclear matrix targeting signal.
Assuntos
Papillomavirus Bovino 1/genética , Proteínas de Ligação a DNA/genética , Genoma Viral , Matriz Nuclear/genética , Proteínas Oncogênicas Virais/genética , Proteínas Virais/genética , Animais , Células CHO , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cricetulus , Proteínas de Ligação a DNA/metabolismo , Repressão Epigenética , Matriz Nuclear/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Virais/metabolismoRESUMO
The efficient transfection of cloned genes into cells has a critical role in nucleic acid-based therapeutic applications, molecular and cell biology studies, and the production of recombinant proteins in cultured cells. Using a stearylated cell-penetrating peptide (CPP) NickFect51, we have generated an effective, universal, and convenient method for the delivery of DNA vectors into animal cells derived from different origins (mammalian, avian, insect). The CPP-mediated transfection described in detail herein is efficient for many regular cell lines commonly used for research purposes and it is especially suitable for transfection of protein production cell lines adapted for growth in chemically defined serum-free medium.
