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Molecular and Cellular Biology, March 2001, p. 1942-1952, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.1942-1952.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Role of the 3' Splice Site in U12-Dependent Intron Splicing

Rosemary C. Dietrich, Marian J. Peris, Andrew S. Seyboldt, and Richard A. Padgett*

Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received 18 October 2000/Returned for modification 22 November 2000/Accepted 13 December 2000

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.

* Corresponding author. Mailing address: Department of Molecular Biology, NC-2, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Phone: (216) 445-2692. Fax: (216) 444-0512. E-mail: padgetr{at}ccf.org.

Molecular and Cellular Biology, March 2001, p. 1942-1952, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.1942-1952.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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