Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro

Abstract

Termination of transcription by RNA polymerase II usually requires the presence of a functional poly(A) site. How the poly(A) site signals its presence to the polymerase is unknown. All models assume that the signal is generated after the poly(A) site has been extruded from the polymerase, but this has never been tested experimentally. It is also widely accepted that a "pause" element in the DNA stops the polymerase and that cleavage at the poly(A) site then signals termination. These ideas also have never been tested. The lack of any direct tests of the poly(A) signaling mechanism reflects a lack of success in reproducing the poly(A) signaling phenomenon in vitro. Here we describe a cell-free transcription elongation assay that faithfully recapitulates poly(A) signaling in a crude nuclear extract. The assay requires the use of citrate, an inhibitor of RNA polymerase II carboxyl-terminal domain phosphorylation. Using this assay we show the following. It has become clear in recent years that RNA polymerase II (RNAPII) not only transcribes the mRNA but shepherds it through the stages of processing as well (42, 67). An early indicator of this coupling between transcription and processing was the finding that the poly(A) signal for cleavage and polyadenylation of pre-mRNA directs not only 3Ј-end processing of the transcript but also termination of transcription by the polymerase (20,32,50,61,77,79). A major challenge has been to explain how the poly(A) signal communicates with the polymerase.The core poly(A) signal in vertebrates consists of two recognition elements flanking a cleavage-polyadenylation site (76,82). Typically, an almost invariant AAUAAA hexamer lies 20 to 50 nucleotides (nt) upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5Ј cleavage product. The cleavage is mediated in vitro by a large, multicomponent protein complex that can be separated into five distinguishable factors. Two of these factors are the cleavage and polyadenylation specificity factor (CPSF), which binds the AAUAAA motif, and the cleavage stimulation factor (CstF), which binds the downstream U-rich element. In vitro studies suggest that CPSF (26) and probably CstF as well (55, 71) are recruited to the polymerase at the promoter. Presumably they then ride with the polymerase during transcription, scanning the extruding transcript so as to snare the poly(A) site when it emerges. The situation in yeast may be similar (8,29). Strictly speaking, the term "poly(A) site" refers only to the point at which cleavage occurs and the poly(A) tail is appended, but we use the term here to refer to the poly(A) signal as a whole when this helps to distinguish between the poly(A) signal as an entity and poly(A) signal transduction [or "poly(A) signaling"] as a process.Models that attempt to explain transduction of the signal from the poly(A) site to the polymerase can be divided into two categories (50): (i) cl...

Functional interactions between the transcription and mRNA 3′ end processing machineries mediated by Ssu72 and Sub1

“…This result may be related to the recent findings using in vitro assays (Tran et al 2001) and EM visualization of RNAP II transcription (Osheim et al 1999(Osheim et al , 2002 to show that cleavage is not a prerequisite for termination in higher eukaryotic cells. Our results support a model in which termination in eukaryotes is dependent on an earlier event, such as recognition of polyadenylation signals by the processing machinery.…”

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

“…This was accomplished using a HeLa nuclear extraction protocol (Tran et al 2001) and verified by immunoblots probed with the CCT1 primary antibody (Fig. 1).…”

“…The HeLa cells were lysed following the HeLa nuclear extraction protocol (Tran et al 2001). Briefly, the pellet was washed with iced phosphate buffer (137 mM NaCl, 2.68 mM KCl, 4.29 mM Na 2 HPO 4 , and 1.47 mM KH 2 PO 4 ).…”

The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity

“…To investigate the basal mechanism of poly(A)-dependent pausing and termination, we placed poly(A) signals into foreign sequence backgrounds, taking particular care to ensure that DNA downstream of the poly(A) signal was devoid of any natural elements that might modify poly(A) signal activity (Tran et al 2001;Orozco et al 2002). It was such experiments that previously showed that polymerase density first rises and then falls after crossing the poly(A) signal, reflecting poly(A)-dependent pausing of the polymerases followed by release (Orozco et al 2002).…”