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 SeñalAsunto(s)
Inhibidores Enzimáticos/farmacología , Cinesinas/antagonistas & inhibidores , Sulfonas/farmacología , Adenosina Trifosfato/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Mitosis/efectos de los fármacos , Estructura Molecular , Interferencia de ARN , Bibliotecas de Moléculas Pequeñas , Sulfonas/químicaRESUMEN
BACKGROUND: The accurate alignment of chromosomes at the spindle equator is fundamental for the equal distribution of the genome in mitosis and thus for the genetic integrity of eukaryotes. Although it is well established that chromosome movements are coupled to microtubule dynamics, the underlying mechanism is not well understood. RESULTS: By combining RNAi-depletion experiments with in vitro biochemical assays, we demonstrate that the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in mammalian tissue culture cells. We show that in vitro Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably, Kif18A like its yeast ortholog Kip3p depolymerizes longer microtubules more quickly than shorter ones. In vivo, Kif18A accumulates in mitosis where it localizes close to the plus ends of kinetochore microtubules. The depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores, resulting in the activation of the Mad2-dependent spindle-assembly checkpoint. Live-cell microscopy studies revealed that in Kif18A-depleted cells, chromosomes move at reduced speed and completely fail to align at the spindle equator. CONCLUSIONS: These studies identify Kif18A as a dual-functional kinesin and a key component of chromosome congression in mammalian cells.
