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Projeto de investigação
Genetic Diseases and RNA Processing
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RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts
Publication . Pacheco, Teresa; Moita, Luis; Gomes, Anita Q.; Hacohen, Nir; Carmo-Fonseca, Maria
U2AF is a heterodimeric splicing factor composed of a large (U2AF) and a small (U2AF) subunit. In humans, alternative splicing generates two U2AF variants, U2AF and U2AF. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF affected the expression level of ∼500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF-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 U2AF or U2AF altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF is essential for HeLa cell division and suggest a novel role for both U2AF protein isoforms as regulators of alternative splicing of a specific subset of genes.
Diversity of vertebrate splicing factor U2AF35 : identification of alternatively spliced U2AF1 mRNAS
Publication . Pacheco, Teresa R.; Gomes, Anita Q.; Barbosa-Morais, Nuno; Benes, Vladimir; Ansorge, Wilhelm; Wollerton, Matthew; Smith, Christopher W.; Valcárcel, Juan; Carmo-Fonseca, Maria
U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U2AF(35)c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF(35)b differs from the previously described U2AF(35)a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF(65). Biochemical experiments indicate that isoform U2AF(35)b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF(65), stimulates U2AF(65) binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF(35)a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF(35)a and U2AF(35)b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF(35) protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.
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Fundação para a Ciência e a Tecnologia
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POCI
Número da atribuição
POCTI/MGI/36547/2000