RESUMEN
Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3' end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3' end processing complex, CF I(m)68, stimulates mRNA export. CF I(m)68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF I(m)68 may act as a novel adaptor for NXF1/TAP, we show that CF I(m)68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF I(m)68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3' end processing factor CF I(m)68 in mRNA export.
Asunto(s)
Procesamiento de Término de ARN 3' , Precursores del ARN/metabolismo , Transporte de ARN , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Centrifugación por Gradiente de Densidad , Células HeLa , Humanos , Carioferinas/metabolismo , Ratones , Modelos Biológicos , Células 3T3 NIH , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Ribosomas/metabolismo , Transcripción Genética , Proteína Exportina 1RESUMEN
Mammalian cleavage factor I (CF Im) is an essential factor that is required for the first step in pre-mRNA 3' end processing. Here, we characterize CF Im68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF Im68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF Im68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF Im68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFIm68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF Im68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3' end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF Im68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus.
Asunto(s)
Estructuras del Núcleo Celular/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencias de Aminoácidos , Animales , Bromouracilo/análogos & derivados , Cromatina/metabolismo , Cromatina/ultraestructura , Diclororribofuranosil Benzoimidazol/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa/efectos de los fármacos , Humanos , Mamíferos , Microscopía Electrónica/métodos , Mutación , Proteínas Nucleares/metabolismo , Fotoblanqueo , Subunidades de Proteína , ARN/metabolismo , ARN Polimerasa II/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Empalme Serina-Arginina , Transcripción Genética , Uridina/análogos & derivados , Uridina/metabolismo , Factores de Escisión y Poliadenilación de ARNm/efectos de los fármacos , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Cleavage factor I(m) (CF I(m)) is required for the first step in pre-mRNA 3'-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68-kDa subunits. The binding of CF I(m) to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF I(m) involved in RNA binding, protein-protein interactions, and subcellular localization. CF I(m)68 has a modular domain organization consisting of an N-terminal RNA recognition motif and a C-terminal alternating charge domain. However, the RNA recognition motif of CF I(m)68 on its own is not sufficient to bind RNA but is necessary for association with the 25-kDa subunit. RNA binding appears to require a CF I(m)68/25 heterodimer. Whereas multiple protein interactions with other 3'-end-processing factors are detected with CF I(m)25, CF I(m)68 interacts with SRp20, 9G8, and hTra2beta, members of the SR family of splicing factors, via its C-terminal alternating charge domain. This domain is also required for targeting CF I(m)68 to the nucleus. However, CF I(m)68 does not concentrate in splicing speckles but in foci that partially colocalize with paraspeckles, a subnuclear component in which other proteins involved in transcriptional control and RNA processing have been found.