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Projeto de investigação
ANÁLISE IN VIVO DA FUNÇÃO DO FACTOR DE SPLICING U2AF NUM MODELO CELULAR DE VERTEBRADO
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In vivo requirement of the small subunit of U2AF for recognition of a weak 3′ splice site
Publication . Pacheco, Teresa; Coelho, Miguel B.; Desterro, Joana; Mollet, Inês G.; Carmo-Fonseca, Maria
The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF(65)) and a small subunit, U2AF(35). U2AF(65) binds to the polypyrimidine tract upstream from the 3' splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF(35) plays a role in assisting U2AF(65) recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3' splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF(65) triggers the downregulation of U2AF(35). We further show that the knockdown of each U2AF subunit inhibits weak 3' splice site recognition, while overexpression of U2AF(65) alone is sufficient to activate the selection of this splice site. A variant of U2AF(65) lacking the interaction domain with U2AF(35) shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3' splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF(35) regulates the selection of weak 3' splice sites in a specific subset of cellular transcripts.
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.
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.
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Fundação para a Ciência e a Tecnologia
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POCTI
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PRAXIS XXI/BD/18044/98