RESUMO
The mRNA of Scamper, a putative intracellular calcium channel activated by sphingosylphosphocholine, contains a long 5' transcript leader with several upstream AUGs. In this work we have investigated the role this sequence plays in the translational control of Scamper expression. The cytosolic transcription machinery of a T7 RNA polymerase recombinant vaccinia virus was used to avoid artifacts arising from cryptic promoters or mRNA processing. Based on transient transfection experiments of dicistronic and bi-monocistronic plasmids expressing reporter genes, we present evidence that the 5' transcript leader of Scamper contains a functional internal ribosome entry site (IRES). Our data indicate that Scamper translation in Madin-Darby canine kidney cells is driven by a cap-independent mechanism supported by the IRES activity of its mRNA. Finally, the Scamper IRES appears to be the first IRES with specificity for kidney epithelial cells.
Assuntos
Canais de Cálcio/genética , Biossíntese de Proteínas/genética , Ribossomos/metabolismo , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Biologia Computacional , DNA Complementar/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Rim/citologia , Rim/metabolismo , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Naturally occurring sphingoid molecules control vital functions of the cell through their interaction with specific receptors. Proliferation, differentiation and programmed death result in fact from a fine balance of signals, among which sphingosine and structurally related molecules play fundamental roles, acting as either first or second messengers. The corresponding receptors need to be identified in order that the role of sphingoid molecules can be established. Among them, several G-protein-coupled receptors specific for sphingosine 1-phosphate, sphingosylphosphocholine, or both, have already been investigated. In contrast, the identification of the postulated intracellular receptors has been problematical. In the present study we re-evaluated the molecular characterization of Scamper, the first proposed intracellular receptor for sphingosylphosphocholine [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1993-1996] and commonly believed to be a Ca(2+) channel of the endoplasmic reticulum (the name "SCaMPER" used by Mao et al. being derived from "sphingolipid Ca(2+)-release-mediating protein of the endoplasmic reticulum"). In contrast with what has been believed hitherto, our primary-structure and overexpression experiments indicate that Scamper is a 110-amino-acid protein spanning the membrane once with a Nexo/Ccyt topology [von Heijne and Gavel (1988) Eur. J. Biochem. 174, 671-678]. Overexpression of either wild-type or tagged Scamper induces a specific phenotype characterized by the rapid extension of actin-containing protrusions, followed by cell death.
