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Vol. 17, Issue 10, 4187-4199, October 2006

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RNA Interference Knockdown of hU2AF³⁵ Impairs Cell Cycle Progression and Modulates Alternative Splicing of Cdc25 Transcripts

Teresa Raquel Pacheco^*, Luís Ferreira Moita^*,, Anita Quintal Gomes^*,, Nir Hacohen, and Maria Carmo-Fonseca^*

*Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal; Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Charlestown, MA 02129; and Departamento de Ciências da Saúde, Universidade da Madeira, 9000-390 Funchal, Portugal

Submitted January 13, 2006; Revised July 10, 2006; Accepted July 11, 2006
Monitoring Editor: Karsten Weis

U2AF is a heterodimeric splicing factor composed of a large (U2AF⁶⁵) and a small (U2AF³⁵) subunit. In humans, alternative splicing generates two U2AF³⁵ variants, U2AF³⁵a and U2AF³⁵b. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF³⁵a isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF³⁵a affected the expression level of 500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF³⁵a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF³⁵a or U2AF³⁵b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF³⁵a is essential for HeLa cell division and suggest a novel role for both U2AF³⁵ protein isoforms as regulators of alternative splicing of a specific subset of genes.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-01-0036) on July 19, 2006.

Address correspondence to: Maria Carmo-Fonseca (carmo.fonseca{at}fm.ul.pt)

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