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J. Biol. Chem., Vol. 282, Issue 4, 2221-2228, January 26, 2007

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Mutations in the SF1-U2AF⁵⁹-U2AF²³ Complex Cause Exon Skipping in Schizosaccharomyces pombe^*

Noriko Haraguchi, Tomoko Andoh, David Frendewey, and Tokio Tani¹

From the Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan and Regeneron Pharmaceuticals, Inc., Tarrytown, New York 10591-6707

To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4⁺ gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4⁺ mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura⁺ mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1⁺, ods2⁺, and ods3⁺ encode splicing factors Prp2p/U2AF⁵⁹, U2AF²³, and SF1, respectively, which form a SF1-U2AF⁵⁹-U2AF²³ complex involved in recognition of the branch-point and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF⁵⁹-U2AF²³ binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF⁵⁹-U2AF²³ complex is essential for ordered exon joining in constitutive splicing in S. pombe.

Received for publication, October 5, 2006 , and in revised form, November 20, 2006.

^* This work was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan (to T. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan. Tel. & Fax: 81-96-342-3461; E-mail: ttani{at}sci.kumamoto-u.ac.jp.

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