title: RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.
abstract: 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 approximately 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.
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors.forename: Teresa Raquel
authors.initials: TR
authors.lastname: Pacheco
authors.forename: Luís Ferreira
authors.initials: LF
authors.lastname: Moita
authors.forename: Anita Quintal
authors.initials: AQ
authors.lastname: Gomes
authors.forename: Nir
authors.initials: N
authors.lastname: Hacohen
authors.forename: Maria
authors.initials: M
authors.lastname: Carmo-Fonseca
mesh_terms: D017398:Alternative Splicing; D017209:Apoptosis; D001483:Base Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics*; D002455:Cell Division; D049109:Cell Proliferation; D016000:Cluster Analysis; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D020033:Protein Isoforms / Q000235:genetics; D034741:RNA, Small Interfering; D012261:Ribonucleoproteins / Q000235:genetics* / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D000072668:Splicing Factor U2AF; D014158:Transcription, Genetic; D014162:Transfection; D020687:cdc25 Phosphatases / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases