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Molecular and Cellular Biology, July 2002, p. 4579-4586, Vol. 22, No. 13
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.13.4579-4586.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of Specific Protein-RNA Complexes Associated with the Coupling of Polyadenylation and Last-Intron Removal

Charles Cooke and James C. Alwine*

Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6142

Received 4 September 2001/ Returned for modification 17 October 2001/ Accepted 26 March 2002

Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (AC', AC, and BC) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The AC' complex is formed by 30 s after mixing, the AC complex is formed between 1 and 2 min after mixing, and the BC complex is formed by 2 to 3 min after mixing. AC' is a precursor of AC, and the AC' and/or AC complex is a precursor of BC. Of the three complexes, BC appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the AC' complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the AC' complex is dependent on the polypyrimidine tract. The transition from AC' to AC appears to require an intact 3'-splice site. Formation of the BC complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the AC' complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the BC complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.

* Corresponding author. Mailing address: 314 Biomedical Research Building, 421 Curie Blvd., University of Pennsylvania, Philadelphia, PA 19104-6142. Phone: (215) 898-3256. Fax: (215) 573-3888. E-mail: alwine{at}mail.med.upenn.edu

Molecular and Cellular Biology, July 2002, p. 4579-4586, Vol. 22, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.13.4579-4586.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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