RESUMEN
Messenger RNA (mRNA) export from the nucleus is essential for eukaryotic gene expression. Here we identify a transcript-selective nuclear export mechanism affecting certain human transcripts, enriched for functions in genome duplication and repair, controlled by inositol polyphosphate multikinase (IPMK), an enzyme catalyzing inositol polyphosphate and phosphoinositide turnover. We studied transcripts encoding RAD51, a protein essential for DNA repair by homologous recombination (HR), to characterize the mechanism underlying IPMK-regulated mRNA export. IPMK depletion or catalytic inactivation selectively decreases RAD51 protein abundance and the nuclear export of RAD51 mRNA, thereby impairing HR. Recognition of a sequence motif in the untranslated region of RAD51 transcripts by the mRNA export factor ALY requires IPMK. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), an IPMK product, restores ALY recognition in IPMK-depleted cell extracts, suggesting a mechanism underlying transcript selection. Our findings implicate IPMK in a transcript-selective mRNA export pathway controlled by phosphoinositide turnover that preserves genome integrity in humans.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Inestabilidad Genómica , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero/genética , Línea Celular Tumoral , Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Humano , Recombinación Homóloga/genética , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Imaging of live cells was carried out using evanescent-wave excitation on a polymer waveguide chip. Integrated waveguide-based interferometric light modulators were fabricated in order to demonstrate on-chip control of excitation light, e.g., for time-lapse fluorescence microscopy. When combined with a sensitive high-resolution imaging system, the integrated waveguide-excitation platform provides an ideal method of near-surface studies of live cells, where photobleaching and/or phototoxicity effects are of critical concern.
Asunto(s)
Imagenología Tridimensional/métodos , Dispositivos Laboratorio en un Chip , Luz , Polímeros/química , Animales , Supervivencia Celular , Adhesiones Focales/metabolismo , Interferometría , Células LLC-PK1 , Microscopía , PorcinosRESUMEN
We present detailed characterization of a unique high-index-contrast integrated optical polymer waveguide platform where the index of the cladding material is closely matched to that of water. Single-mode waveguides designed to operate across a large part of the visible spectrum have been fabricated and waveguide properties, including mode size, bend loss and evanescent coupling have been modeled using effective-index approximation, finite-element and finite-difference time domain methods. Integrated components such as directional couplers for wavelength splitting and ring resonators for refractive-index or temperature sensing have been modeled, fabricated and characterized. The waveguide platform described here is applicable to a wide range of biophotonic applications relying on evanescent-wave sensing or excitation, offering a high level of integration and functionality. The technology is biocompatible and suitable for wafer-level mass production.
