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Molecular and Cellular Biology, November 2001, p. 7673-7681, Vol. 21, No. 22
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.22.7673-7681.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing

Sabine Guth,1 Thomas Ø. Tange,1,dagger Esther Kellenberger,2 and Juan Valcárcel1,*

Gene Expression Programme1 and Structural Biology Programme,2 European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Received 30 July 2001/Accepted 27 August 2001

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF65) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF35) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF35 and the 3' splice site AG can stabilize U2AF65 binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF35 has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF65 binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF65. In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF35 dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF35 dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF35 function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF35 activity and because a truncation mutant of U2AF35 consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF35 can be explained in terms of enhanced U2AF65 binding, other activities of U2AF35 do not correlate with increased cross-linking of U2AF65 to the Py tract. Collectively, the results argue that interaction of U2AF35 with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF65 binding, thus revealing a dual function for U2AF35 in pre-mRNA splicing.

* Corresponding author. Mailing address: Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Phone: 49-6221-387 156. Fax: 49-6221-387 306. E-mail: juan.valcarcel{at}embl-heidelberg.de.

dagger Present address: Department of Biochemistry, Brandeis University, Waltham, Mass.

Molecular and Cellular Biology, November 2001, p. 7673-7681, Vol. 21, No. 22
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.22.7673-7681.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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