Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing. Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified. This study describes another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I. This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15. PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa. Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15. The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15. Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation. Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody (Amrani, 1997).

The direct association between messenger RNA (mRNA) 3'-end processing and the termination of transcription was established for the CYC1 gene of Saccharomyces cerevisiae. The mutation of factors involved in the initial cleavage of the primary transcript at the poly(A) site (RNA14, RNA15, and PCF11) disrupts transcription termination at the 3' end of the CYC1 gene. In contrast, the mutation of factors involved in the subsequent polyadenylation step (PAP1, FIP1, and YTH1) had little effect. Thus, cleavage factors link transcription termination of RNA polymerase II with pre-mRNA 3'-end processing (Birse, 1998).

Cleavage and polyadenylation of mRNA 3' ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, it has been shown that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein-protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1 (Gross, 2001a).

In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. This study shows that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system (Gross, 2001b).

The RNA polymerase II CTD is essential for 3' end cleavage of metazoan pre-mRNAs and binds 3' end processing factors in vitro. This study shows genetic and biochemical interactions between the CTD and the Pcf11 subunit of the yeast cleavage/polyadenylation factor, CFIA. In vitro binding to Pcf11 required phosphorylation of the CTD on Ser2 in the YSPTSPS heptad repeats. Deletion of the yeast CTD reduced the efficiency of cleavage at poly(A) sites, and the length of poly(A) tails suggesting that it helps couple 3' end formation with transcription. Consistent with this model, the 3' end processing factors CFIA, CFIB, and PFI were recruited to genes progressively, starting at the 5' end, in a process that required ongoing transcription (Licatalosi, 2002).

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre-mRNA 3' end processing, binds to the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. The conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3' end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3' end processing activity of Pcf11p and a deficiency of Pcf11p in 3' end processing did not prevent CTD binding. Transcriptional run-on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. It is concluded that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination (Sadowski, 2003).

During transcription, RNA polymerase (Pol) II synthesizes eukaryotic messenger RNA. Transcription is coupled to RNA processing by the carboxy-terminal domain (CTD) of Pol II, which consists of up to 52 repeats of the sequence Tyr 1-Ser 2-Pro 3-Thr 4-Ser 5-Pro 6-Ser 7. After phosphorylation, the CTD binds tightly to a conserved CTD-interacting domain (CID) present in the proteins Pcf11 and Nrd1, which are essential and evolutionarily conserved factors for polyadenylation-dependent and -independent 3'-RNA processing, respectively. This study describes the structure of a Ser 2-phosphorylated CTD peptide bound to the CID domain of Pcf11. The CTD motif Ser 2-Pro 3-Thr 4-Ser 5 forms a beta-turn that binds to a conserved groove in the CID domain. The Ser 2 phosphate group does not make direct contact with the CID domain, but may be recognized indirectly because it stabilizes the beta-turn with an additional hydrogen bond. Iteration of the peptide structure results in a compact beta-spiral model of the CTD. The model suggests that, during the mRNA transcription-processing cycle, compact spiral regions in the CTD are unravelled and regenerated in a phosphorylation-dependent manner (Meinhart, 2004).

The C-terminal domain (CTD) of the large subunit of RNA polymerase II is a platform for mRNA processing factors and links gene transcription to mRNA capping, splicing and polyadenylation. Pcf11, an essential component of the mRNA cleavage factor IA, contains a CTD-interaction domain that binds in a phospho-dependent manner to the heptad repeats within the RNA polymerase II CTD. The phosphorylated CTD exists as a dynamic disordered ensemble in solution and, by induced fit, it assumes a structured conformation when bound to Pcf11. In addition, cis-trans populations were detected for the CTD prolines, and it was found that only the all-trans form is selected for binding. These data suggest that the recognition of the CTD is regulated by independent site-specific modifications (phosphorylation and proline cis-trans isomerization) and, probably, by the local concentration of suitable binding sites (Noble, 2005).

In Saccharomyces cerevisiae, the cleavage/polyadenylation factor Pcf11 is an important regulatory factor required for recruiting the polyadenylation machinery to the elongating RNA polymerase II (RNAPII) and is necessary for correct transcriptional termination. The interaction with RNAPII is mediated by a CTD-interacting domain (CID) located in the N-terminal region of Pcf11 that binds in a phospho-dependent manner the heptad repeats in the RNAPII CTD. This study examineds the interaction of the CID with different RNA sequences and looks at the effect of phosphopeptides derived from the CTD heptad repeats on the RNA-protein interaction. The findings demonstrate that the CID displays weak RNA-binding activity, but with some degree of sequence preference, the RNA-protein and peptide-protein interfaces overlap and the CTD-derived phosphopeptides and RNA compete for the binding site. It is proposed that competition between the protein-peptide and the protein-RNA interaction is important mechanistically and required for the disengagement of polyadenylation factors from RNAPII (Hollingworth, 2006).

The torpedo model of transcription termination by RNA polymerase II proposes that a 5'-3' RNA exonuclease enters at the poly(A) cleavage site, degrades the nascent RNA, and eventually displaces polymerase from the DNA. Cotranscriptional degradation of nascent RNA has not been directly demonstrated, however. This study reports that two exonucleases, Rat1 and Xrn1, both contribute to cotranscriptional degradation of nascent RNA, but this degradation is not sufficient to cause polymerase release. Unexpectedly, Rat1 functions in both 3'-end processing and termination by enhancing recruitment of 3'-end processing factors, including Pcf11 and Rna15. In addition, the cleavage factor Pcf11 reciprocally aids in recruitment of Rat1 to the elongation complex. These results suggest a unified allosteric/torpedo model in which Rat1 is not a dedicated termination factor, but is an integrated component of the cleavage/polyadenylation apparatus (Luo, 2006).

CTD-dependent dismantling of the RNA polymerase II elongation complex by the yeast pre-mRNA 3'-end processing factor, Pcf11

Pcf11 is one of numerous proteins involved in pre-mRNA 3'-end processing and transcription termination. Using elongation complexes (ECs) formed from purified yeast RNA polymerase II (Pol II), it is shown that a 140-amino acid polypeptide from yeast Pcf11 is capable of dismantling the EC in vitro. This action depends on the C-terminal domain (CTD) of the largest subunit of Pol II and the CTD-interaction domain (CID) of Pcf11. These experiments reveal a novel termination mechanism whereby Pcf11 bridges the CTD to the nascent transcript and causes dissociation of both Pol II and the nascent transcript from the DNA in the absence of nucleotide hydrolysis. It is posited that conformational changes in the CTD are transduced through Pcf11 to the nascent transcript to cause termination (Zhang, 2005).

Transcription termination in eukaryotes is essential for recycling polymerase II (Pol II) and for preventing Pol II from perturbing promoters of genes located downstream from a transcription unit (Proudfoot, 2002,; Proudfoot 2004). The mechanism of Pol II termination is poorly understood. Termination depends on the polyadenylation site in the nascent transcript. Numerous proteins involved in cleavage and polyadenylation also appear to participate in termination since mutations in several of these proteins affect both RNA processing and termination. Termination also depends on the C-terminal domain (CTD) of the largest subunit of Pol II (McCracken, 1997; Park, 2004). The CTD is composed of repeating heptapeptide motifs with the consensus YSPTSPS, and undergoes cycles of phosphorylation and dephosphorylation during the transcription process (Buratowski, 2003). It functions as both a binding site and an allosteric regulator of the RNA processing machinery and serves to couple RNA processing and transcription elongation (Bentley, 2002; Zhang, 2005 and references therein).

In addition to the termination that occurs at the end of a transcription unit, premature termination within the body of genes can affect the processivity of Pol II in ways that regulate gene expression. One of the clearest examples of this is HIV. In the absence of the virally encoded protein Tat, transcription from the LTR results in accumulation of short transcripts in the cytoplasm due to premature termination. HIV Tat modifies the elongation complex (EC) by recruiting the kinase, P-TEFb (Price, 2000; see Cyclin dependent kinase 9). P-TEFb phosphorylates the CTD of Pol II, and phosphorylation of the CTD increases the processivity of Pol II at least in part by counteracting the inhibitory action of two elongation factors, NELF and DSIF (Wada, 1998; Yamaguchi, 1999, 2002; Renner, 2001). Several studies have provided evidence that cellular activators stimulate gene expression by increasing the processivity of Pol II, although it is not clear if this occurs by preventing premature termination or by reducing the frequency of pauses and arrests (Zhang, 2005).

Isolated Pol II ECs are remarkably stable, resisting disruption by high salt, sarkosyl, heparin, and even proteinase K. This raises the question of how the EC is dismantled from the DNA. Moreover, how does the CTD impinge on the processivity since the CTD projects from the body of the Pol II molecule in an unstructured conformation and is attached by a flexible linker (Meinhart, 2004; Noble, 2005)? The negative elongation factors NELF and DSIF slow the rate of elongation but do not appear to dissociate the EC (Wada, 1998; Yamaguchi, 1998, 1999). Nucleosomes also inhibit elongation, but they alone do not appear to induce termination. A protein called TTF2 (also known as factor 2) disrupts both Pol II ECs in an ATP-dependent fashion, but this protein appears to function during mitosis to clear ECs from the DNA during chromosome condensation. Moreover, since TTF2 also acts upon Pol I, its function does not appear to be through the CTD (Zhang, 2005 and references therein).

An antibody interacting with the CTD of Drosophila Pol II has been shown to caused Pol II to release the nascent transcript (Zhang, 2004). A search for a Drosophila protein that had similar activity resulted in the identification of a protein with sequence similarity to yeast Pcf11. Mutations in yeast Pcf11 cause Pol II to read through a transcription terminator in vivo (Sadowski, 2003). Moreover, Pcf11 associates with the CTD, and a defect in termination correlates with mutations in the CTD-interacting domain (CID) of Pcf11 (Sadowski, 2003). Since genetic data implicated Pcf11 in Pol II termination in yeast, the effect yeast Pcf11 has on an EC formed from purified yeast Pol II was examined. A novel CTD-dependent mechanism for dismantling the EC was identified and it is proposed that Pcf11 is the engine that drives some termination reactions (Zhang, 2005).

Pcf11 dismantles a Pol II EC by a mechanism that involves the CTD of Pol II. Pcf11 associates with the CTD, in accordance with previous reports (Barilla, 2001; Licatalosi, 2002; Sadowski, 2003). Pcf11 binds RNA, thus allowing Pcf11 to form a bridge between the CTD and the nascent transcript. It is speculated that this bridge transmits force generated by conformational changes occurring at the CTD to the nascent transcript. The force exerted on the nascent transcript could then disrupt the 8-nt heteroduplex formed between the 3'-end of the nascent transcript and the DNA template within the EC, thus resulting in total dissolution of the EC (Kireeva, 2000; Komissarova, 2002). This mechanism is analogous to the mechanism by which Rho is thought to disrupt ECs in bacteria (Richardson 2002), but with an important distinction: Pcf11 dismantles the EC in the absence of nucleotide hydrolysis. The Pcf11-mediated reaction is not unique to the yeast proteins used in this study: The same reaction occurs when Drosophila Pcf11 is incubated with ECs formed from purified Drosophila Pol II (Zhang, 2005).

The discovery of this Pcf11-mediated reaction offers a new perspective on how the CTD of Pol II might regulate transcription elongation. The CTD is required for terminating transcription at the ends of genes. In yeast, Pcf11 is part of a complex called CF1 that is involved in cleaving and polyadenylating pre-mRNA (Gross, 2001a). In vivo cross-linking analyses show that Pcf11 is concentrated in the vicinity of the polyadenylation signal relative to the rest of the transcription unit (Kim, 2004a). CF1 recognizes part of the polyadenylation signal in the nascent transcript (Gross, 2001b), thus providing a way to recruit Pcf11 to the EC at a point that would be appropriate for termination. It is proposed that Pcf11 is the engine that disrupts the EC. In accord with this, a three-amino acid mutation in Pcf11 previously shown to impair termination in vivo (Sadowski, 2003), also abrogates Pcf11's dismantling activity. Characterization of multiple alleles of Pcf11 has provided evidence that Pcf11 contains functionally distinct domains (Sadowski, 2003). Mutations in the CID impair termination, while mutations elsewhere in the protein impair the cleavage and polyadenylation reactions. Importantly, some alleles that impair the RNA processing reactions do not impair termination. This suggests that cleavage of the nascent transcript during the polyadenylation reaction is not essential for termination (Zhang, 2005).

Recruitment of Pcf11 by the polyadenylation signal provides a mechanism by which polyadenylation and termination can be coupled. Termination occurs downstream from the polyadenylation signal, possibly in a stochastic manner. It is proposed that Pcf11 is transferred from the polyadenylation signal in the nascent transcript to the EC, where it becomes poised to trigger transcript release. Evidence suggests that termination is preceded by pausing, and analysis of the dismantling reaction with Drosophila Pol II and Pcf11 indicates that Pcf11 acts only on paused Pol II. This fits the model for the dismantling reaction. When Pol II is undergoing active elongation, RNA reeling out of the Pol II will inhibit the transfer of force from the CTD to the heteroduplex buried inside the EC (Zhang, 2005).

An alternative mechanism for coupling polyadenylation and termination involves an RNA exonuclease that initiates degradation at the uncapped 5'-end of the nascent transcript generated when the polyadenylation machinery cleaves the transcript. An important role for this reaction in yeast is indicated by the widespread impairment of termination observed in a Rat1 mutant strain (Kim, 2004b). Rat1 is a 5'-to-3' exonuclease that is thought to chase down the EC as it degrades the uncapped nascent transcript. However, it seems unlikely that Rat1 alone can cause termination, since ECs remain intact even after nascent transcripts have been extensively degraded with various ribonucleases. It is proposed that Rat1 facilitates the action of Pcf11. By shortening the transcript, Rat1 could reduce the distance between Pcf11's point of contact on the nascent transcript and the RNA exit channel. A short distance between these two points would be necessary if force generated by conformational changes in the CTD is to impact on the nascent transcript within the EC. The Rat1 homolog Xrn1 might function to facilitate Pcf11 in human cells as well (Zhang, 2005).

The CTD also controls the elongation properties of Pol II as it clears the promoter and traverses the body of the gene. Efficient elongation correlates with hyperphosphorylation of the CTD. In vivo cross-linking analysis shows that Pcf11 is present throughout transcription units in yeast, albeit at a level lower than found in the vicinity of the polyadenylation signal (Kim, 2004a). Given its distribution on the gene, Pcf11 could be poised to induce premature termination under circumstances that cause Pol II to pause. Hence, Pcf11 might be a new target that is counteracted by the action of activators and elongation factors involved in promoting transcriptional elongation in vivo (Zhang, 2005).

Mammalian Pcf11

Six different protein factors are required in vitro for 3' end formation of mammalian pre-mRNAs by endonucleolytic cleavage and polyadenylation. Five of the factors have been purified and most of their components cloned, but cleavage factor II(m) (CF II(m)) has remained uncharacterized. CF II(m) was purified from HeLa cell nuclear extract by several chromatographic steps. During purification, CF II(m) activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIB(m)) for the cleavage reaction. CF IIA(m) fractions contain the human homologs of two yeast 3' end processing factors, Pcf11p and Clp1p, as well as cleavage factor I(m) (CF I(m)) and several splicing and transcription factors. hClp1 and is a genuine subunit of CF IIA(m). Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract. hClp1 interacts with CF I(m) and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3' end processing factors within the cleavage complex (de Vries, 2000).

Pcf11: Biological Overview | Regulation | Developmental Biology | References

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