InteractiveFly: GeneBrief

Protein 1 of cleavage and polyadenylation factor 1: Biological Overview | References

The mechanism by which Pol II terminates transcription in metazoans is not understood. This study shows that Pcf11 is directly involved in termination in Drosophila. dPcf11 is concentrated at the 3' end of the hsp70 gene in cells, and depletion of dPcf11 with RNAi causes Pol II to readthrough the normal region of termination. dPcf11 also localizes to most transcribed loci on polytene chromosomes. Biochemical analysis reveals that dPcf11 dismantles elongation complexes by a Pol II C-terminal domain (CTD) dependent but nucleotide-independent mechanism and that dPcf11 forms a bridge between the CTD and RNA. This bridge appears to be crucial because an anti-CTD antibody, which also dismantles the elongation complex, is found to bridge the CTD to RNA. dPcf11 was observed to inhibit transcription at low, but not high, nucleotide levels, suggesting that dPcf11 dismantles paused elongation complexes. These results provide a biochemical basis for the dependency of termination on pausing and the CTD in metazoans (Zhang, 2006).

Termination of Pol II transcription is an essential step in gene expression, but the mechanism is poorly understood. Besides its requirement for recycling use of Pol II, the choice of termination site can influence the availability of splice sites and polyadenylation sites in pre-mRNA. Half of the mRNAs in humans utilize alternate polyadenylation sites, and this can affect the location, stability, and coding potential of the transcripts. Pol II molecules that fail to terminate can inhibit function of downstream promoters by displacing proteins from the DNA. This so-called transcription interference can serve to regulate expression of some genes (Zhang, 2006 and references therein).

Pol II termination is coupled to polyadenylation by the polyadenylation signal in the nascent transcript. Two models have been proposed to explain this coupling. According to the torpedo model, cleavage of the nascent transcript, which precedes polyadenylation, generates an uncapped end on the residual transcript engaged with Pol II. This uncapped end is an entry point for a 5' to 3' exonuclease that chases down the Pol II and induces termination. The torpedo model received recent support with the finding that mutation of a 5' to 3' exonuclease, called Rat1, causes Pol II to readthrough terminators in yeast (Kim, 2004b). Depletion of the homologous protein Xrn2 from human cells also impairs termination on a transiently transfected β globin gene (Zhang, 2006 and references therein).

An alternative model, originally called the antiterminator model but now generalized as the allosteric model, posits that the polyadenylation signal in the nascent transcript causes an allosteric change in Pol II that decreases the processivity of the elongation complex (EC). This could be due to the dissociation of an antiterminator from the EC or association of a factor that depresses processivity. Until recently, the strongest support for the allosteric model was provided by circumstances in which termination occurs in the absence of the cleavage reaction. Under these circumstances, the torpedo model for termination cannot apply, as there is no entry point for the 5' to 3' exonuclease (Zhang, 2006 and references therein).

Recently, a yeast protein called Pcf11 dismantles a yeast Pol II EC (Zhang, 2005). This reaction depends on the CTD of Pol II, thus providing a possible reason for why deletion of the CTD impairs termination in human cells (McCracken, 1997). The CTD corresponds to the unusual C-terminal domain of the largest Pol II subunit and is composed of multiple copies of a heptapeptide with the consensus YSPTSPS (Buratowski, 2003; Meinhart, 2005). yPcf11 appears to dismantle the EC by bridging the CTD to the nascent transcript (Zhang, 2005). In yeast, mutations in yPcf11 impair both termination and polyadenylation (Sadowski, 2003). yPcf11 is in a complex called CF1A, which is involved in processing the 3' end of mRNAs (Gross, 2001a). CF1A recognizes part of the tripartite polyadenylation signal in the GAL7 gene (Gross, 2001b), thus providing a possible basis for how the polyadenylation signal might recruit or regulate the activity of yPcf11. Human Pcf11 is in a complex with at least 15 other polypeptides, and the complex is required for 3' end processing in vitro (de Vries, 2000). The hPcf11 complex interacts with CF1m and CPSF, two proteins that recognize the polyadenylation signal in the nascent transcript (de Vries, 2000; Venkataraman, 2005). Nothing is known about the role of hPcf11 in termination (Zhang, 2006 and references therein).

Given the results that termination can occur prior to nascent transcript cleavage, and the discovery that yPcf11 could be the engine that drives the termination reaction in yeast (Zhang, 2005), it was asked whether Pcf11 is involved in termination in Drosophila (Zhang, 2006).

This study provides evidence that dPcf11 is directly involved in Pol II termination. Immunofluorescence microscopy and ChIP indicate that dPcf11 is concentrated at the 3' end of the hsp70 gene, and depletion of dPcf11 from Drosophila cells increases the level of Pol II normally detected downstream from the polyadenylation signal of hsp70. In addition, the N-terminal region of dPcf11 completely dismantles an elongation complex. This last result sets dPcf11 apart from all other proteins that have been implicated in Pol II termination and is strong evidence that dPcf11 is directly involved in termination. The detection of dPcf11 at most highly transcribed loci in polytene chromosomes suggests that dPcf11 is involved in termination at many genes. dPcf11 provides a basis for connecting three key aspects of termination: the CTD, the polyadenylation signal, and pausing (Zhang, 2006).

A crucial step in the termination reaction mediated by dPcf11 appears to be the formation of a bridge between the CTD and the nascent transcript, since this is the only functional aspect common to the CTD antibody and dPcf11, both of which dismantled the EC. Additional support for the importance of the bridge comes from the analysis of yeast Pcf11: mutations impairing RNA binding or CTD binding each inhibit the dismantling reaction (Zhang, 2005). In addition, the dismantling reaction can be inhibited by hybridizing a DNA oligonucleotide to the nascent transcript in the region just outside from where RNA exits Pol II (Zhang, 2005). Presumably, the oligonucleotide blocks formation of the bridge by interfering with the Pcf11-RNA interaction (Zhang, 2006).

Because the CTD antibody and Pcf11 are structurally unrelated, it is unlikely that the dismantling reaction involves Pcf11 directly recognizing part of the body of Pol II. How the formation of the bridge disrupts the elongation complex is a mystery. One possibility is that constraining the CTD or the RNA causes either of these or Pcf11 itself to contact the RNA exit channel in a way that destabilizes the EC. RNA-protein interactions in the RNA exit channel of bacterial RNA polymerase contribute to pausing and termination (Toulokhonov, 2003). The molecular contacts at the RNA exit channel of the Pol II EC may be uniquely suited for allosteric control of the EC, because it was observed that Rho, which normally functions in termination in bacteria, disrupts Pol II ECs, but not Pol I or Pol III ECs (Lang, 1998). Rho moves along RNA in a 5' to 3' direction, so it probably collides with the region of Pol II at the RNA exit channel (Zhang, 2006).

dPcf11 seems to interact with a relatively small region of the Drosophila CTD. This is in contrast with the yeast and human CTDs where Pcf11 could in principal coat almost all of the yeast CTD and half of the human CTD. The results from Drosophila suggest that the bridge does not have to form close to the body of the Pol II molecule to dismantle the EC. The binding of dPcf11 to the Drosophila CTD may not be dictated by the heptad per se but by a slightly larger motif that appears four times in the region where dPcf11 bound the CTD. This motif, PSYSPTSP, corresponds to the region of a peptide composed of two consensus heptads that was contacted by yPcf11 in a crystallized complex (Meinhart, 2004; Zhang, 2006).

Phosphorylation of the CTD could influence the activity of Pcf11. Phosphorylation of serine 2 in the CTD appears to increase the affinity of yPcf11 for the CTD (Barilla, 2001, Licatalosi, 2002 and Sadowski, 2003). Importantly though, yPcf11 binds the unphosphorylated CTD, and there is evidence in yeast indicating that the CTD of Pol II is dephosphorylated just prior to termination (Buratowski, 2005). ChIP data indicate that the level of serine 2 phosphorylation increases as Pol II moves from the 5′ to the 3′ end of the hsp70 gene (Boehm, 2003), and the same occurs on several yeast genes (Ahn, 2004). This rising level of serine 2 phosphorylation could contribute to the recruitment of Pcf11 near the 3′ end of the gene. However, the phosphates on the CTD might also antagonize the ability of Pcf11 to form a bridge with the nascent transcript due to electrostatic repulsion. The CTD phosphatase Ssu72 has been implicated in termination (Krishnamurthy, 2004: Steinmetz, 2003). Ssu72 might participate in termination by removing phosphates from the CTD so the bridge can form between the CTD and RNA (Zhang, 2006).

These results show that dPcf11 is concentrated near the polyadenylation signal of hsp70, similar to what was observed for several genes in yeast (Kim, 2004a). Though Pcf11 binds RNA, it seems unlikely that Pcf11 alone recognizes the polyadenylation signal in the nascent transcript for two reasons: (1) equivalent crosslinking is observed to two unrelated RNAs, neither of which contained a polyadenylation signal; (2) amino acids 1-149 of Pcf11 lack any known RNA recognition motifs. Nevertheless, Pcf11 appears to have a surface that interacts specifically with RNA, because mutating one amino acid in yeast Pcf11 impaired RNA binding without affecting CTD binding (Zhang, 2005; Zhang, 2006).

Yeast Pcf11 is part of a complex called CF1A, which contains three other subunits (Gross, 2001a). One subunit, Rna15, recognizes part of the polyadenylation signal, thus providing a way to recruit yPcf11 to the end of the gene after the polyadenylation signal has been transcribed (Gross, 2001b). Human Pcf11 is part of a complex called CFIIA, which itself does not appear to recognize the polyadenylation signal (de Vries, 2000). CFIIA, however, interacts with CPSF and CFI_m, two proteins that recognize different parts of the polyadenylation signal in humans and that are involved in pre-mRNA 3' end processing. If CPSF and CFI_m are involved in recruiting Pcf11 to the 3' end of genes in metazoans, regulation is needed to prevent Pcf11 from prematurely terminating transcription. ChIP detects both CPSF and CFI_m well upstream of the polyadenylation site in the human G6PD gene), and earlier studies indicated that CPSF could be recruited to the 5' end of genes through association with TFIID (Zhang, 2006 and references therein).

The location of pause sites will be a key parameter in dictating where Pcf11 dismantles the elongation complex. As long as the EC is moving, it resists the action of Pcf11. It is suspected that this resistance arises because the RNA reeling out of an actively moving EC interferes with physical interactions that might be required for the dismantling reaction. There are ample data to indicate that pause sites are involved in selection of termination sites (Plant, 2005 and references therein). Diverse mechanisms could be used by the cell to cause the EC to pause. These include the presence of pause sites that are intrinsic to the DNA sequence (Palangat, 2004). Intrinsic pauses are found scattered throughout almost any stretch of DNA, so this could account for the stochastic selection of termination sites downstream from a polyadenylation signal. Specific proteins bound to the DNA could cause pausing as appears to be the case for the MAZ protein. Finally, nucleosomes cause ECs to pause. This could explain why chromatin remodeling factors appear to act as terminators (Zhang, 2006 and references therein).

It is concluded that dependence of the Pcf11 dismantling reaction on pausing and the CTD provide possible explanations for why these two things are important for termination. The specificity of termination probably arises from the combinatorial actions of factors that control pausing, the association of Pcf11 with the CTD, and the association of Pcf11 with the nascent transcript. These results provide direct support for an allosteric model of termination but certainly do not preclude possible contributions from an RNA exonuclease after cleavage of the nascent transcript. One possibility that has been proposed is that the exonuclease shortens the residual nascent transcript, forcing Pcf11 to bind close to the RNA exit channel (Zhang, 2005; Zhang, 2006).

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. This study demonstrates that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination (Kamieniarz-Gdula, 2019).

Search PubMed for articles about Drosophila Pcf11

Ahn, S. H., Kim, M. and Buratowski, S. (2004). Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing, Mol. Cell 13: 67-76. PubMed ID: 14731395

Barilla, D., Lee. B. A. and Proudfoot, N. J. (2001). Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. 98: 445-450. PubMed ID: 11149954

Boehm, A. K., Saunders, A., Werner, J. and Lis, J. T. (2003). Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol. 23(21): 7628-37. PubMed ID: 14560008

Buratowski, S., (2003). The CTD code. Nat. Struct. Biol. 10: 679-680. PubMed ID: 12942140

Buratowski, S. (2005). Connections between mRNA 3' end processing and transcription termination. Curr. Opin. Cell Biol. 17: 257-261. PubMed ID: 15901494

de Vries, H., et al. (2000). Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors, EMBO J. 19: 5895-5904. PubMed ID: 11060040

Gross, S. and Moore, C. (2001a). Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I. Proc. Natl. Acad. Sci. 98: 6080-6085. PubMed ID: 11344258

Gross, S. and Moore, C. L. (2001b). Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation, Mol. Cell. Biol. 21: 8045-8055. PubMed ID: 11689695

Kamieniarz-Gdula, K., Gdula, M. R., Panser, K., Nojima, T., Monks, J., Wisniewski, J. R., Riepsaame, J., Brockdorff, N., Pauli, A. and Proudfoot, N. J. (2019). Selective roles of vertebrate PCF11 in premature and full-length transcript termination. Mol Cell 74(1): 158-172. PubMed ID: 30819644

Kim, M., et al. (2004a). Transitions in RNA polymerase II elongation complexes at the 3' ends of genes. EMBO J. 23: 354-364. PubMed ID: 14739930

Kim, M., et al. (2004b). The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432: 517-522. PubMed ID: 15565157

Krishnamurthy, S., et al. (2004). Ssu72 is an RNA polymerase II CTD phosphatase, Mol. Cell 14: 387-394. PubMed ID: 15125841

Lang, W. H., Platt, T. and Reeder, R. H. (1998). Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III, Proc. Natl. Acad. Sci. 95: 4900-4905. PubMed ID: 9560200

Licatalosi, D. D., et al. (2002). Functional interaction of yeast pre-mRNA 3' end processing factors with RNA polymerase II. Mol. Cell 9: 1101-1111. PubMed ID: 12049745

McCracken, S., et al. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385: 357-361. PubMed ID: 9002523

Meinhart, A., et al. (2005). A structural perspective of CTD function, Genes Dev. 19: 1401-1415. PubMed ID: 15964991

Palangat, M., Hittinger, C. T. and Landick, R. (2004). Downstream DNA selectively affects a paused conformation of human RNA polymerase II. J. Mol. Biol. 341: 429-442. PubMed ID: 15276834

Plant, K. E., et al. (2005). Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Ggamma-globin gene, Mol. Cell. Biol. 25: 3276-3285. PubMed ID: 15798211

Sadowski, M., et al. (2003). Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination. EMBO J. 22: 2167-2177. PubMed ID: 12727883

Steinmetz, E. J. and Brow, D. A. (2003). Ssu72 protein mediates both poly(A)-coupled and poly(A)-independent termination of RNA polymerase II transcription. Mol. Cell. Biol. 23: 6339-6349. PubMed ID: 12944462

Toulokhonov, I. and Landick, R. (2003). The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination. Mol. Cell 12: 1125-1136. PubMed ID: 14636572

Venkataraman, K., Brown, K. M. and Gilmartin, G. M. (2005). Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition. Genes Dev. 19: 1315-1327. PubMed ID: 15937220

Zhang, Z., Fu, J. and Gilmour, D. S. (2005). CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3'-end processing factor, Pcf11. Genes Dev. 19: 1572-80. PubMed ID: 15998810

Zhang, Z. and Gilmour, D. S. (2006). Pcf11 is a termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript. Mol. Cell 21(1): 65-74. PubMed ID: 16387654

date revised: 10 December 2020

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