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Molecular and Cellular Biology, March 2004, p. 1855-1869, Vol. 24, No. 5
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.5.1855-1869.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

An Intronic Enhancer Regulates Splicing of the Twintron of Drosophila melanogaster prospero Pre-mRNA by Two Different Spliceosomes

Petra Scamborova, Anthony Wong, and Joan A. Steitz*

Department of Molecular Biophysics and Biochemistry, Yale University Howard Hughes Medical Institute, New Haven, Connecticut 06536-9812

Received 12 September 2003/ Returned for modification 28 October 2003/ Accepted 18 November 2003

We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5' splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.

* Corresponding author. Mailing address: Department of Molecular Biophysics and Biochemistry, Yale University, Howard Hughes Medical Institute, 295 Congress Avenue, New Haven, CT 06536-9812. Phone: (203) 737-4418. Fax: (203) 737-4418. E-mail: joan.steitz{at}yale.edu.

Molecular and Cellular Biology, March 2004, p. 1855-1869, Vol. 24, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.5.1855-1869.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Borah, S., Wong, A. C., Steitz, J. A. (2009). Drosophila hnRNP A1 homologs Hrp36/Hrp38 enhance U2-type versus U12-type splicing to regulate alternative splicing of the prospero twintron. Proc. Natl. Acad. Sci. USA 106: 2577-2582 [Abstract] [Full Text]  
  • Brock, J. E., Dietrich, R. C., Padgett, R. A. (2008). Mutational analysis of the U12-dependent branch site consensus sequence. RNA 14: 2430-2439 [Abstract] [Full Text]  
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