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J. Biol. Chem., Vol. 280, Issue 27, 25478-25484, July 8, 2005

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Substrate-dependent Differences in U2AF Requirement for Splicing in Adenovirus-infected Cell Extracts^*

Martin Lützelberger, Ellenor Backström, and Göran Akusjärvi

From the Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden

U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF⁶⁵ subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF³⁵ subunit contacts the 3'-splice site AG. Here we show that U2AF³⁵ appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF³⁵ for splicing in nuclear extracts from uninfected cells, are transformed to U2AF³⁵-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.

Received for publication, December 7, 2004 , and in revised form, April 25, 2005.

^* This work was supported by the Swedish Cancer Society and the Wallenberg Consortium North. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Figs. 1 and 2.

Present address: Dept. of Molecular Biology, University of Aarhus, C.F. Møllers Alle, Bldg. 130, DK-8000 Aarhus C, Denmark.

To whom correspondence should be addressed: Dept. of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, Husargatan 3, Box 582, 75123 Uppsala, Sweden. Tel.: 46-18-4714164; Fax: 46-18-509876: E-mail: goran.akusjarvi{at}imbim.uu.se.

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