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Vol. 18, Issue 4, 1282-1292, April 2007

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Subnuclear Localization and Dynamics of the Pre-mRNA 3' End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

Stefano Cardinale^*,, Barbara Cisterna, Paolo Bonetti^*, Chiara Aringhieri^*, Marco Biggiogera, and Silvia M.L. Barabino^*

*Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milan, Italy; and Department of Animal Biology, Laboratory of Cell Biology and Neurobiology, and Institute of Molecular Genetics, Consiglio Nazionale delle Ricerche, University of Pavia, 27100 Pavia, Italy

Submitted September 22, 2006; Revised January 3, 2007; Accepted January 22, 2007
Monitoring Editor: Karsten Weis

Mammalian cleavage factor I (CF I_m) is an essential factor that is required for the first step in pre-mRNA 3' end processing. Here, we characterize CF I_m68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF I_m68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF I_m68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF I_m68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFI_m68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF I_m68 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 I_m68 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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, United Kingdom.

Address correspondence to: Silvia M.L. Barabino (silvia.barabino{at}unimib.it)

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