The RNA tether from the poly(A) signal to the polymerase mediates coupling of transcription to cleavage and polyadenylation

Abstract

We have investigated the mechanism by which transcription accelerates cleavage and polyadenylation in vitro. By using a coupled transcription-processing system, we show that rapid and efficient 3' end processing occurs in the absence of crowding agents like polyvinyl alcohol. The continuity of the RNA from the poly(A) signal down to the polymerase is critical to this processing. If this tether is cut with DNA oligonucleotides and RNaseH during transcription, the efficiency of processing is drastically reduced. The polymerase is known to be an integral part of the cleavage and polyadenylation apparatus. RNA polymerase II pull-down and immobilized template experiments suggest that the role of the tether is to hold the poly(A) signal close to the polymerase during the early stages of processing complex assembly until the complex is sufficiently mature to remain stably associated with the polymerase on its own.