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Molecular and Cellular Biology, February 2009, p. 1072-1082, Vol. 29, No. 4
0270-7306/09/$08.00+0     doi:10.1128/MCB.01071-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Spliceosome Assembly Pathways for Different Types of Alternative Splicing Converge during Commitment to Splice Site Pairing in the A Complex{triangledown} ,{dagger}

Matthew V. Kotlajich, Tara L. Crabb, and Klemens J. Hertel*

Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697-4025

Received 9 July 2008/ Returned for modification 22 August 2008/ Accepted 24 November 2008

Differential splice site pairing establishes alternative splicing patterns resulting in the generation of multiple mRNA isoforms. This process is carried out by the spliceosome, which is activated by a series of sequential structural rearrangements of its five core snRNPs. To determine when splice sites become functionally paired, we carried out a series of kinetic trap experiments using pre-mRNAs that undergo alternative 5' splice site selection or alternative exon inclusion. We show that commitment to splice site pairing in both cases occurs in the A complex, which is characterized by the ATP-dependent association of the U2 snRNP with the branch point. Interestingly, the timing of splice site pairing is independent of the intron or exon definition modes of splice site recognition. Using the ATP analog ATP{gamma}S, we showed that ATP hydrolysis is required for splice site pairing independent from U2 snRNP binding to the pre-mRNA. These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025. Phone: (949) 824-2127. Fax: (949) 824-8598. E-mail: khertel{at}uci.edu

{triangledown} Published ahead of print on 8 December 2008.

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

Molecular and Cellular Biology, February 2009, p. 1072-1082, Vol. 29, No. 4
0270-7306/09/$08.00+0     doi:10.1128/MCB.01071-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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