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

Abstract

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 (A C , A C , and B C ) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A C complex is formed by 30 s after mixing, the A C complex is formed between 1 and 2 min after mixing, and the B C complex is formed by 2 to 3 min after mixing. A C is a precursor of A C , and the A C and/or A C complex is a precursor of B C . Of the three complexes, B C 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 A C complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A C complex is dependent on the polypyrimidine tract. The transition from A C to A C appears to require an intact 3-splice site. Formation of the B C 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 A C complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B C 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.

Association of polyadenylation cleavage factor I with U1 snRNP

“…The subunits also sediment with very large complexes that appear to represent polyadenylation complexes or complexes associated with the coupling of polyadenylation and splicing (Cooke and Alwine 2002).…”

“…Indeed, U1 snRNP is known to be released from the splicesome after U4, U5, and U6 entry and prior to catalysis. Thus the successful coupling of polyadenylation and splicing, resulting in the removal of the last intron (Cooke et al 1999;Cooke and Alwine 2002), would release U1 snRNP, along with polyadenylation factors with which it interacted. Given the established interdependence of last intron removal and polyadenylation, it is interesting to consider that the release of U1snRNP may signal that spliceosome formation has been successful, providing a positive signal for polyadenylation.…”

“…It has also been proposed that poly(A) polymerase contributes to the coupling of splicing and polyadenylation through interactions with the splicing factor U2AF bound to the polypyrimidine tract in the last intron (Vagner et al 2000). Recently an RNA-protein complex associated with coupling has been identified (Cooke and Alwine 2002). This coupling complex appears to be made up of splicosomal and polyadenylation complex proteins.…”

“…The coupling of the two processing reactions is also integrally linked to exon definition (Niwa et al 1990a;Robberson et al 1990;Niwa and Berget 1991a,b;Berget 1995), particularly the definition of the last exon of a pre-mRNA, which involves protein-protein interactions across the exon between splicing factors at the 3Ј splice site and components of the polyadenylation complex at the polyadenylation signal (Niwa et al 1990a,b;Niwa and Berget 1991a,b;Scott and Imperiale 1996;Cooke et al 1999;Cooke and Alwine 2002). Studies from this laboratory and others have identified a number of such interactions.…”

Split genes and their expression in Kaposi's sarcoma‐associated herpesvirus

“…An internal exon is also usually defined as less than 500 nts in size (51). RNA 5′ capping (100, 101) and 3′ polyadenylation (102,103) couple with RNA splicing to promote recognition of terminal splice sites. Negative effects of KSHV ORF57 and PAN RNA on RNA splicing are hypothetical.…”