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.