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Molecular and Cellular Biology, November 2006, p. 8183-8190, Vol. 26, No. 21
0270-7306/06/$08.00+0     doi:10.1128/MCB.00350-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Requirement of the Small Subunit of U2AF for Recognition of a Weak 3' Splice Site ^,

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal

Received 27 February 2006/ Returned for modification 29 March 2006/ Accepted 8 August 2006

The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF⁶⁵) and a small subunit, U2AF³⁵. U2AF⁶⁵ 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 U2AF³⁵ plays a role in assisting U2AF⁶⁵ 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 U2AF⁶⁵ triggers the downregulation of U2AF³⁵. We further show that the knockdown of each U2AF subunit inhibits weak 3' splice site recognition, while overexpression of U2AF⁶⁵ alone is sufficient to activate the selection of this splice site. A variant of U2AF⁶⁵ lacking the interaction domain with U2AF³⁵ 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, U2AF³⁵ regulates the selection of weak 3' splice sites in a specific subset of cellular transcripts.

Published ahead of print on 28 August 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

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