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In this review, recent progress relating to the integration of nuclear events …
Biology Articles » Molecular Biology » RNA polymerase II and the integration of nuclear events » Polyadenylation and the CTD
Polyadenylation of mRNA takes place in two steps: endonucleolytic cleavage of the mRNA precursor followed by poly(A) addition to the 3' end of the upstream cleavage product. mRNA polyadenylation requires multiple protein factors, including, in mammalian cells cleavage/polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), two cleavage factors, CFI and CFII, and poly(A) polymerase (for review, see Colgan and Manley 1997; Zhao et al. 1999a). Early studies showing that transcription termination by RNAP II was dependent on an intact poly(A) signal (for review, see Proudfoot 1989) predicted a possible link between the transcription process and polyadenylation, and there is now solid evidence that these two processes are indeed intimately coupled (see Fig. 4).
As with capping and splicing, RNAs transcribed by CTD-truncated RNAP II were not efficiently polyadenylated in transiently transfected cells (McCracken et al. 1997b). It was also shown that CPSF and CstF present in unfractionated nuclear extracts could bind GST-CTD, and both were present in an RNAP II holoenzyme preparation. It appears that the 50 kD subunit of the heterotrimeric CstF (CstF-50) interacts directly with the CTD, based on binding experiments with GST-CTD and in vitro translated proteins. Unlike capping and splicing, the phosphorylation status of the CTD was found not to affect binding.
An initial interpretation of these results was that the CTD functions to help recruit polyadenylation factors to sites of RNAP II transcription, increasing their local concentration and thereby facilitating efficient processing. But how and when do poly(A) factors associate with the polymerase? The initially unexpected (but perhaps not so surprising in light of the capping and splicing connections described above) answer is that at least some of the action occurs at the promoter. While studying the general transcription factor TFIID, Dantonel et al. (1997) found that an extensively purified preparation contained in good yield at least three of the four subunits of CPSF. Furthermore, in a reconstituted transcription assay, CPSF was shown to transfer from TFIID to RNAP II concomitant with initiation. Together with the results of McCracken et al. (1997b), these findings suggest that at least some factors associate early with RNAP II and remain associated with it during elongation.
The above findings were extended by a biochemical study demonstrating a more direct role of RNAP II in polyadenylation (Hirose and Manley 1998). These authors provided evidence that RNAP II, and specifically the CTD, is required for the cleavage reaction in vitro in the absence of transcription. Purified RNAP IIA and II0 were both found to activate the first step of polyadenylation, 3' cleavage, in a reconstituted system containing all the other polyadenylation factors. In addition, both unphosphorylated and hyperphosphorylated GST-CTD proteins activated cleavage just as efficiently as did RNAP II, although in this case the hyperphosphorylated CTD was more active than the nonphosphorylated form. In addition, 3' cleavage in nuclear extracts could be inhibited by immunodepletion of RNAP II and rescued by add-back of the purified enzyme. These results suggest that the CTD of RNAP II participates directly in the formation and/or function of a stable, catalytically active processing complex through direct interaction with polyadenylation factor(s).
The factors required for mRNA polyadenylation have been intensively studied for over a decade. How had the involvement of RNAP II in 3' processing escaped attention? Hirose and Manley (1998) suggest that it was masked by an apparent requirement for the small molecule creatine phosphate (CP) in the cleavage reaction. CP is usually employed as part of an ATP regeneration system for ATP requiring reactions and has been routinely added to 3'-processing reactions, where conflicting early experiments had suggested a possible ATP requirement. But Hirose and Manley (1997) showed that CP plays a different role in 3' cleavage: ATP was found in fact not to be required for cleavage (at least in mammals), but CP seemed to be, although hydrolysis of its high-energy bond does not occur. These and other results led to the hypothesis that CP functions by mimicking a phosphoprotein, and this in turn resulted in the discovery that RNAP II0 can activate cleavage in the absence of CP (Hirose and Manley 1998). If indeed CP and the CTD function similarly, this has significant implications regarding mechanism. It would disfavor a model in which the CTD functions to facilitate complex assembly by making contacts with two or more factors, and favor an allosteric activation model. Or perhaps it does both, which seems to be the case in capping (see above). But the CP-CTD connection is not perfect. For example, the concentration of CP required to support optimal cleavage is ~107 fold greater than the required CTD concentration, and the CP-CTD model does not offer an explanation for why unphosphorylated CTD can activate cleavage.
The direct involvement of RNAP II in 3' cleavage could indicate that the reaction must occur very shortly after the poly(A) site is transcribed, or that RNAP II might play an essential but transient role, perhaps facilitating formation of a conformation necessary for processing. Some support for the former hypothesis comes from studies examining cotranscriptional polyadenylation using a coupled in vitro transcription-polyadenylation system (Yonaha and Proudfoot 1999). Specific G-rich sequences originally identified as protein binding sites and proposed to contribute to transcription termination in vivo were found to promote RNAP II pausing in vitro and, importantly, to significantly enhance polyadenylation at an upstream poly(A) site in a transcription-dependent manner. A plausible explanation for these findings is that pausing the polymerase near the site of processing allows more time for the CTD to function in cleavage before RNAP II moves downstream or terminates (see Fig. 4).
Another recent study extended further the potential significance of the association between polyadenylation factors and RNAP II into the realm of DNA repair and tumor suppressors (Kleiman and Manley 1999). These authors discovered in a yeast two-hybrid screen an interaction between Cst-50 (the CTD-interacting subunit of CstF; see above) and BRCA1-associated ring domain protein (BARD1). BRCA1 is a breast/ovarian cancer-associated tumor suppressor protein of unknown function, although it has been suggested to be associated with the RNAP II holoenzyme (Scully et al. 1997a) and to participate in the response to DNA damage (Scully et al. 1997b). BARD1 is tightly associated with BRCA1 in vivo (Wu et al. 1996) and likely contributes to its function. CstF-50 and BARD1 were found to associate in vitro and a fraction of intact CstF is associated with the BARD1/BRCA1 complex in vivo. In functional assays, recombinant BARD1 was found to inhibit the 3'-cleavage reaction in vitro. Given the apparent association of BARD1/BRCA1 and CstF with elongating RNAP II, an intriguing model is that the function of the BARD1 inhibitory interaction is to prevent premature polyadenylation of nascent RNAs at sites of RNAP II pausing, for example, at sites of DNA damage.
The intimate coupling between transcription and polyadenylation mediated by the CTD is likely a general feature in mammalian cells, as we have just seen. Does it also occur in yeast cells? When the S. cerevisiae Cup1 gene was transcribed by a CTD-deleted RNAP II, 3' processing of the Cup1 transcripts became inefficient (McNeil et al. 1998). On the other hand, yeast Cyc1 and Yhr54C transcripts made by CTD-deleted RNAP II (McNeil et al. 1998), as well as His4 transcript made by RNAP I (Lo et al. 1998), were efficiently polyadenylated in vivo. These observations indicate that the CTD-mediated coupling between transcription and polyadenylation might be a gene specific feature in yeast. Nonetheless, additional studies have indicated that the CTD could mediate a physical and mechanistic link between transcription and polyadenylation in yeast. First, the yeast polyadenylation factor Pta1, which may function as an assembly factor (Zhao et al. 1999b), as has been suggested for a related human protein, symplekin (Takagaki and Manley 2000), could be selected from unfractionated yeast extract by phosphorylated GST-CTD (Rodriguez et al. 2000). These authors also showed that kin28 mutant alleles defective for CTD phosphorylation resulted in a reduction in Pta1 levels. A second intriguing finding originated from a genetic screen for new transactivating factors involved in mRNA 3'-end formation. The screen uncovered mutations in an essential gene, Ess1, which encodes a peptidylprolyl-cis-trans-isomerase (PPIase), that led to a defect in 3'-end formation of a plasmid-derived pre-mRNA (Hani et al. 1999). Importantly, Ess1 has recently been found to associate specifically and directly with the hyperphosphorylated CTD (Morris et al. 1999). We will discuss Ess1 and other PPIases in the next section in relation to their possible functions in remodeling CTD-associated protein complexes. But together these results support the existence of an RNAP II CTD-polyadenylation link in yeast as well as mammals.
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