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Projeto de investigação
Regulation of mRNA processing and transport within the nucleus
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Frontiers in fluorescence microscopy
Publication . Rino, José; Braga, José; Henriques, Ricardo; Carmo-Fonseca, Maria
How we see organisms and cells depends on the tools at our disposal. For over 150 years, biologists were forced to rely on fixed, dehydrated and stained specimens in order to guess how the living cells could function. It all changed abruptly during the last two decades when the rapid development of novel imaging techniques revolutionized the way scientists look at the structures of life alive.
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.
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Fundação para a Ciência e a Tecnologia
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POCI
Número da atribuição
POCI/SAU-MMO/57700/2004