Functional Coupling of Last-Intron Splicing and 3′-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage

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Abstract
We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3' splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3'-end processing of this exon can be distinguished. First, the 3' splice site establishes connections to enhance 3'-end processing, while the nascent 3'-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3'-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3' end, and then finishing with splicing of the last intron.