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

doi:10.1128/MCB.21.22.7673-7681.2001

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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^, 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-kDasubunit of U2AF (U2AF⁶⁵) binds to the polypyrimidine (Py) tract preceding the 3' splicesite, while the 35-kDa subunit (U2AF³⁵) contacts the conserved AG dinucleotide at the 3' end of theintron. 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-dependentpre-mRNAs. U2AF³⁵ has also been implicated in arginine-serine (RS) domain-mediatedbridging interactions with splicing factors of the SR proteinfamily bound to exonic splicing enhancers (ESE), and these interactionscan also stabilize U2AF⁶⁵ binding. Complementation of the splicing activity of nuclearextracts depleted of U2AF by chromatography in oligo(dT)-celluloserequires, for some pre-mRNAs, only the presence of U2AF⁶⁵. In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2pre-mRNA requires both U2AF subunits. In this report we have investigatedthe sequence elements (e.g., Py tract strength, 3' splice siteAG, ESE) responsible for the U2AF³⁵ dependence of IgM. The results indicate that (i) the IgM substrateis an AG-dependent pre-mRNA, (ii) U2AF³⁵ dependence correlates with AG dependence, and (iii) the identityof the first nucleotide of exon 2 is important for U2AF³⁵ function. In contrast, RS domain-mediated interactions with SRproteins bound to the ESE appear to be dispensable, because thepurine-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 andlacking 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 interactionof U2AF³⁵ with a consensus 3' splice site triggers events in spliceosomeassembly in addition to stabilizing U2AF⁶⁵ binding, thus revealing a dual function for U2AF³⁵ in pre-mRNAsplicing.

^* Corresponding author. Mailing address: Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1,69117 Heidelberg, Germany. Phone: 49-6221-387 156. Fax: 49-6221-387306. E-mail: juan.valcarcel{at}embl-heidelberg.de.

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

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