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Nucleic Acids Research, 2001, Vol. 29, No. 8 1741-1749
© 2001 Oxford University Press

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Gil Ast, Thomas Pavelitz1 and Alan M. Weiner1,*

Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel and 1Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350, USA

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem–loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem–loop IIa can participate in spliceosome assembly.

* To whom correspondence should be addressed. Tel: +1 206 543 1768; Fax: +1 206 685 9231; Email: amweiner{at}u.washington.edu


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