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Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome

¹ Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; ² Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA; ³ Howard Hughes Medical Institute and Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56’s ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF⁶⁵ and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56’s dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF⁶⁵. Our results indicate that hUAP56 first interacts with U2AF⁶⁵ in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.

[Keywords: hUAP56; splicing; prespliceosome assembly; spliceosome assembly; ATP binding; ATPase; RNA helicase]

Received January 30, 2008; revised version accepted May 9, 2008.

E-MAIL michael.green{at}umassmed.edu; FAX: (508) 856-5473.

⁵ E-MAIL haihongshen{at}gist.ac.kr; FAX: 82-62-970-2484.

Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1657308.

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