Coupling of transcription termination to RNAi

ARTICLE IN PRESS

Journal of Theoretical Biology 245 (2007) 278–289 www.elsevier.com/locate/yjtbi

Coupling of transcription termination to RNAi M. Bahman Bahramian Impedagen, LLC, 175 Brushy Plain Road, Suite 3-C6, Branford, CT 06405-2617, USA Received 22 April 2006; received in revised form 21 October 2006; accepted 25 October 2006 Available online 29 October 2006

Abstract In metazoans, the mechanisms of transcriptional termination by RNA polymerase II (Pol II) and accelerated decay of messenger RNA (mRNA) following transcription shutdown are linked by sharing the same sequence elements and mRNA elongation, processing and termination factors. This begs the question, how could one process have two opposite outcomes, making or degrading mRNA? An integrated ‘‘allosteric-GENEi-torpedo’’ model that could explain this paradox predicts participation of two novel factors: (1) An allosteric factor, regulated by a physiological repressor, binds to a unique sequence element of a gene near the site of cleavage and polyadenylation, poly(A) site, and acts on the homologous site on the nascent transcript to cause its cleavage. The conformational changes of this factor determine the fate of nascent RNA, either to get cleaved and processed to mature mRNA for directing protein synthesis, or not to get cleaved and become template for double-stranded (ds) RNA synthesis. (2) A general transcription termination factor, recruited by transcribing Pol II at the poly(A) site, allostrically alters and induces Pol II to switch template from DNA to nascent RNA several hundred nucleotides downstream of the poly(A) site. The template switch disengages Pol II from DNA and effectively terminates transcription. The Pol II with newly acquired RNA-dependent RNA polymerase activity retraces its path, back along the nascent RNA, so generating dsRNA. The extent to which it can retrace this path is determined by the factors influencing the cleavage of the pre-mRNA at the site of polyA addition. If cleavage and polyadenylation occur, the retracing is cut short, the 30 RNA is degraded by an exonuclease and the polymerase is liberated to reinitiate transcription. If the cleavage is inhibited, then a full-length dsRNA can be produced. This can then be subject to cleavage by ‘‘Dicer’’, which generates fragments of 22 bp that guide degradation of the cognate mRNA via the RNA interference (RNAi) pathway. This model complements the current ‘‘allosteric-torpedo’’ model of transcription termination, and could explain the apparent paradox of the divergent results of a common biological process. r 2006 Elsevier Ltd. All rights reserved. Keywords: Transcriptional termination; Allosteric model; Torpedo model; Gene impedance (GENEi); RNA interference (RNAi)

1. Introduction Genetic information encoded in the sequence of DNA double helix is transcribed by RNA polymerase II (Pol II) to messenger RNA (mRNA). The transcribed code is carried to the protein-synthesizing machinery defining the sequence of amino acids and, therefore, the structure and function of proteins. Much is known about how transcription begins, but the exact mechanism by which Pol II terminates transcription in metazoans is unclear. Proper transcriptional termination is critical for successful gene expression. First, it allows release of transcripts from Pol Tel.: +1 203 315 2932; fax: +1 925 891 3518.

E-mail address: [email protected]. 0022-5193/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtbi.2006.10.026

II. Second, it allows release of Pol II from template DNA, facilitating its recycling for further rounds of transcription. Third, it ensures that regulations of downstream genes on the same chromosome are not perturbed by read-through polymerases that have failed to terminate at upstream genes. Studies in yeast have demonstrated the occurrence of gene loops in transcriptionally active genes, where a gene’s termination region can be physically linked to its promoter region. Thus, termination can additionally serve to facilitate transcriptional reinitiation (Ansari and Hampsey, 2005; O’Sullivan et al., 2004). In eukaryotic protein-encoding genes, a defining feature of the mRNAs is a 30 end extension of 100–250 adenosine nucleotides, known as the poly(A) tail. This is not encoded by the gene but added following cleavage of the nascent

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transcript. A discrete signal in the DNA that might define the termination site could not be identified. However, the site of cleavage in most pre-mRNAs lies between the highly conserved poly(A) addition signal (AAUAAA) and a downstream sequence element (DSE), which is a U- or GU-rich motif. Cleavage itself occurs predominantly at a CA dinucleotide (Proudfoot, 1989). In mammals, the poly(A) signal is positioned 10–30 nucleotides (nt) upstream of the cleavage site, and the DSE is positioned 10–30 nt downstream of the cleavage site. The Pol II continues transcribing the nascent RNA for another 200–2000 nucleotides beyond the poly(A) site before cleavage occurs and transcription is aborted (Bauren et al., 1998; Dye and Proudfoot, 1999; Osheim et al., 1999). In the two-step pre-mRNA 30 end processing reaction, transcription of the poly(A) signal induces the endonucleolytic cleavage of the nascent transcript, generating an upstream cleavage product that is immediately polyadenylated (Colgan and Manley, 1997; Zhao et al., 1999). The downstream cleavage product, with an uncapped phosphate at its 50 end, is highly unstable and is rapidly degraded (Manley et al., 1982). Molecular factors that recognize the cleavage site, and cut the RNA, bind to a regulatory region of the transcribing Pol II, the carboxy-terminal domain (CTD) of its largest subunit. This interaction is important for recruiting the factors cotranscriptionally to the nascent transcript. In turn, these factors must be off-loaded from the polymerase onto the RNA at their site of action for transcription to be terminated (Ahn et al., 2004; Buratowski, 2003; Kim et al., 2004a). CTD, having a repeated consensus YSPTSPS structure (25 or 26 tandem repeats in yeast, 52 in mammals), is regulated by phosphorylation throughout the transcription cycle, and is believed to coordinate events involved in the production of an mRNA, including pre-mRNA processing steps (for a review, see Zorio and Bentley, 2004). Two multisubunit protein complexes recognize the cis elements and catalyse the cleavage and polyadenylation reactions. In mammals, the 160 kDa subunit of the cleavage-polyadenylation specificity factor (CPSF) has been shown to interact with the poly(A) site, while three other subunits (CPSF-100, CPSF-73, and CPSF-30) are thought to contribute to the specificity and strength of binding. The DSE represents a platform for the interaction with the cleavage stimulatory factor (CstF) via its 64 kDa subunit. The binding of CstF and CPSF appears to be cooperative in that CstF binding to the DSE greatly enhances the affinity of CPSF to the poly(A) site and vice versa. Two additional multisubunit factors, cleavage factor I (CF I) and cleavage factor II (CF II), are essential to direct cleavage of the pre-mRNA. Poly(A) polymerase (PAP) is usually required for the cleavage reaction and together with CPSF directs poly(A) addition. Poly(A) binding protein PABP II binds the emerging poly(A) tail, stabilizing it, and in turn enhances the processivity of the PAP. Most of the components of these complexes have

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homologues in yeast cleavage-polyadenylation apparatus, comprising of the cleavage-polyadenylation factor (CPF) and cleavage factors CFIA and CFIB. Transcript cleavage at the poly(A) site is a key determinant in transcriptional termination. However, a number of detailed nascent transcription studies on several genes have shown that mRNA 30 end processing is not the only requirement for transcription termination. Evidence has been obtained for the requirement of both specific termination factors and sequence elements located downstream of the poly(A) site (Proudfoot et al., 2002, for a review). It has been known for sometime that termination is functionally linked to cleavage and polyadenylation of the nascent transcript’s 30 end (Zaret and Sherman, 1982), and that cleavage-polyadenylation-directing signals are also the cis elements required for normal termination in mammals (Connelly and Manley, 1988; Logan et al., 1987; Whitelaw and Proudfoot, 1986) and in yeast (Russo, 1995). Moreover, a number of yeast cleavage-polyadenylation factors, but not the ones involved solely in polyadenylation, are also needed for proper termination, including CFIA components Rna14, Rna15 and Pcf11 (Birse et al., 1998; Sadowski et al., 2003; Kim et al., 2004a) and CPF subunits Yhh1 (Dichtl et al., 2002) and Ssu72 (Ganem et al., 2003). In order to explain the mechanistic connection between termination and cleavage-polyadenylation by Pol II, two models have been offered, the ‘‘allosteric’’ and ‘‘torpedo’’ models. According to the allosteric model, also known as anti-terminator model, transcription of the poly(A) signal triggers conformational changes in the Pol II elongation complex (EC), for example, by recruitment of a negative elongation factor or release of an anti-termination factor, producing termination competent Pol II (Logan et al., 1987). An alternative ‘‘torpedo’’ model postulated that cleavage of the transcript at the poly(A) site provides an unprotected 50 end of the 30 RNA product, still tethered to the elongating polymerase, which is rapidly degraded by a 50 -30 exonuclease. When the nuclease reaches the polymerase, somehow destabilizes the EC, causing termination (Connelly and Manley, 1988). This model was proposed based on an analogy with bacterial transcription termination, where a Rho factor, an RNA helicase, travels along the downstream cleavage product and destabilizes the elongating RNA polymerase, causing termination (Nudler and Gottesman, 2002). The results of the studies on Pol II transcriptional termination carried out over the past decade have indicated that these two models are not mutually exclusive and termination likely involves aspects of both models. The 50 -30 exonucleases that degrade the downstream RNA cleavage product are necessary but not sufficient for termination. Something in addition is required, and neither the allosteric nor the torpedo model in its simplest form can explain what actually provokes the Pol II to detach itself from the template. Therefore, new integrated models have been proposed. In this regard, a hybrid model proposes that the 50 -30 exonuclease Rat1 in yeast is an essential component of a Pol II complex that

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YEAST Gene

Poly(A) site

Transcription

TRU

Recruitment of mRNA cleavage & polyadenylation factors + DNA-bound TRU at Poly(A) site, and loading of TTU on the CTD of Pol II occur during termination of transcription.

Pol II

m7G

CTD

Nascent mRNA

TTU mRNA cleavage and polyadenylation factors

A

mRNA

AAAAAn Poly(A)

RdRP

5’ exonuclease

B

Cofactor(s)

The 5’ exonuclease loaded on the 5’ end of the 3’ RNA digests the ssRNA template of the RdRP, liberating the polymerase and effecting termination.

AAAAAn

C Complex of TRU and repressor

Dicer

D

TTU-induced conversion of Pol II to RdRP (depicted by amber to blue color shift), switching template from DNA to RNA and initiation of dsRNA synthesis are followed by cleavage and polyadenylation of nascent RNA .

dsRNA

Allosteric change to TRU during gene repression prevents cleavage of the nascent transcript at the poly(A) site, resulting in dsRNA synthesis that leads to RNAi.

RNAi

Fig. 1. Depiction of a switch between transcriptional termination and posttranscriptional silencing of a gene in a lower eukaryote such as yeast. (A) Transcription of a gene by RNA polymerase II produces nascent mRNA, which eventually have two distinctive features: a 7-methyl-guanosine cap (m7G) at the 50 end and a polyadenosine (poly(A)) tail at the 30 end. The carboxy-terminal domain (CTD) of the polymerase comprises repeats of a seven-aminoacid motif and undergoes reversible phosphorylations as the polymerase moves along, recruiting various transcription factors. The nascent mRNA will be cut at the normal site of poly(A) addition. (B) Cleavage at poly(A) site involves, in addition to the known mRNA processing factors, the putative genespecific transcriptional repression unit (TRU). Following cleavage of the poly(A) site, the liberated nascent RNA is polyadenylated that becomes mRNA. TRU remains associated with DNA. A putative general factor recruited by the Pol II CTD at the poly(A) site is designated transcription termination unit (TTU). TTU migrates to the active site of the polymerase and alters its template preference from DNA to RNA. The polymerase switches from DNA template to the nascent RNA and continues adding complementary nucleotides to the already attached 30 end, which results in production of doublestranded (ds) RNA. However, the 50 exonuclease (known as Rat1 in yeast), aided by its cofactors (Rai1 and Rtt103), having loaded onto the free end of the 30 RNA, begins digesting the RNA rapidly and moving toward the polymerase. (C) When the exonuclease eventually catches up to the elongation complex, destabilizes it and causes termination of transcription. (D) Inactivation of TRU by interaction with a physiological repressor (intrinsic GENEi), or its removal by homologous transgenes (alternative GENEi), prevents cleavage at the poly(A) site. Under such condition Rat1 cannot load on the nascent RNA, but the template switching mediated by TTU takes place, and antisense-RNA synthesis on RNA template continues past the poly(A) site, yielding dsRNA in the form of a hairpin, or unlinked dsRNA if an endonuclease cleaves the loop. The resulting dsRNA is subsequently cleaved by a dsRNA endonuclease (Dicer) to short-interfering RNAs (siRNAs) that initiate posttranscriptional gene silencing, better known as RNA interference (RNAi).

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281

HUMAN Poly(A) site TRU

Gene

CoTC site

Transcription Recruitment of mRNA cleavage & polyadenylation factors + DNA-bound TRU at Poly(A) site, and loading of TTU on Pol II CTD occur during transcription termination.

Pol II

m7G

CTD

Nascent mRNA

TTU mRNA cleavage and polyadenylation factors

A

RNA

RdRP

5’

B

mRNA

AAAAAn Poly(A)

5’ exonuclease

3’ ribonuclease 5’

AAAAAn

C Complex of TRU and repressor

Dicer

D

RNAi

dsRNA

TTU-induced conversion of Pol II to RdRP (amber to blue shift), DNA to RNA template switch, and initiation of dsRNA synthesis are followed by cleavage at CoTC site and cleavage plus polyadenylation at the poly(A) site of the nascent RNA. The RNA fragment just 3’ to the poly(A) site is degraded by 3’ RNase. The 5’ribonuclease degrades the CoTC-cleaved distal 3’ RNA, and liberates the polymerase. Allosteric change of TRU during gene repression prevents cleavage of the nascent transcript at the poly(A) site, resulting in dsRNA synthesis that triggers RNAi.

Fig. 2. Depiction of a switch between transcriptional termination and posttranscriptional silencing of a gene in a higher eukaryote such as human b-globin gene. (A) In higher eukaryotes, similar to lower eukaryotes, Pol II produces nascent mRNA with eventually two distinctive features, an m7G cap at the 50 end and a poly(A) tail at the 30 end. The CTD of the Pol II recruits various transcription factors as it moves along. The nascent mRNA will be cut at two positions: at the normal site of poly(A) addition and within the self-cleaved co-transcriptional cleavage site (CoTC). Cleavage at poly(A) site involves, in addition to the known mRNA processing factors, the putative transcriptional repression unit (TRU), which remains associated with a specific DNA sequence. A transcriptional termination unit (TTU) is also loaded on the CTD of Pol II at the poly(A) site that migrates to the catalytic part of the polymerase. (B) As the TTU migrates to the active site of Pol II and alters its template preference from DNA to RNA, the polymerase switches to the nascent RNA and proceeds with generating dsRNA. A newly synthesized CoTC site on the nascent RNA, gets cleaved auto-catalytically. A 50 exonuclease (called Xrn2 in human cells) is loaded onto the free 50 end of the RNA at CoTC site and begins digesting the RNA rapidly and moving toward the polymerase. Following cleavage of the poly(A) site, the liberated nascent RNA is polyadenylated that becomes mRNA. (C) When Xrn2 runs into the polymerase, destabilizes it and terminates transcription. The RNA fragment generated by the cleavages at the poly(A) and CoTC sites, is digested by a 30 exonuclease. (D) Inactivation of TRU by interaction with a physiological repressor (intrinsic GENEi), or removal by homologous transgenes (alternative GENEi), would prevent cleavage at the poly(A) site. Under these conditions transcription of RNA on RNA template continues past the poly(A) site producing dsRNA, provided that cleavage at the CoTC occurs after the polymerase has switched template to the nascent RNA and has transcribed passed the CoTC site. Dicer subsequently cleaves the resulting dsRNA to siRNAs that initiate RNAi.

achieves cleavage at the poly(A) site, degradation of the nascent downstream RNA, and undergoes allosteric changes that promote its release from the template (Luo et al., 2006). But what these allosteric changes might be and

how they would affect dissociation of the EC remain unclear (Rosonina et al., 2006). Previously, we reported, when rodent fibroblast cultured cell lines were transiently transfected with a large number

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of plasmids, each carrying a full-length pro-a1(I) collagen gene, the nuclear localization of such transcriptionally silent transgenes caused strong inhibition of transcription of the cellular pro-a1(I) collagen gene and active degradation of its cognate mRNA by a post-transcriptional gene silencing mechanism (PTGS) (Bahramian and Zarbl, 1999). We have explained this observation within the context of a cellular process that we have defined as gene impedance (GENEi), which involves synthesis of sequence-specific double-stranded RNA (dsRNA) originating in the 30 noncoding region of the nascent transcript (Bahramian and Zarbl, 2005a). According to that theory, synthesis of the dsRNA occurs as part of the normal process of transcriptional termination by Pol II and transcriptional repression. The participation of an RNA-dependent RNA polymerase activity in the dsRNA synthesis was predicted, but the exact mechanism of this action and its connection to the mechanism of transcription termination was unknown (Bahramian and Zarbl, 2005b). Here, a proposed integrated ‘‘allosteric-GENEi-torpedo’’ model (see Figs. 1 and 2) could explain the paradox of one process with two opposite end results, namely, transcription termination versus gene impedance. According to the model, conformational change to a single genespecific allosteric factor, termed TRU (for transcription repression unit), is the key determinant for engagement of Pol II in the alternative tracks of transcription termination versus ‘‘coupled termination and GENEi’’. It is postulated that TRU binds to a sequence element of a gene close to its poly(A) cleavage site, and is required for cleavage of its homologous nascent transcript at the poly(A) site. Another prediction of this model is conversion of the DNA-dependent RNA polymerase II to RNA-dependent RNA polymerase over a stretch of several hundred nucleotides downstream of the poly(A) site. This conversion is mediated by a general transcription termination unit (TTU) that could be a component of the RNA 30 end processing and termination complex. Allosteric changes to the TRU, induced by interaction with a physiological repressor molecule, determine the fate of the nascent transcript of Pol II, either to get cleaved at the poly(A) site and follow the mRNA-processing track to become a complete mRNA, or not to get cleaved and serve as a template for anti-sense RNA synthesis by the de novo TTUinduced RNA-dependent RNA polymerase activity. In the latter case, the resulting dsRNA then undergo processing by ribonuclease III (Dicer) to fragments of 22 bp, which trigger sequence-specific degradation of the cognate mRNA. 2. Torpedo model Studies on yeast and human genes have implicated a 50 30 exonuclease in Pol II termination of transcription, therefore, provided support for the ‘‘torpedo’’ model (Kim et al., 2004b; Teixeira et al., 2004; West et al., 2004). According to this model the nascent RNA is cut while it is

still being synthesized. The liberated 50 region forms the mRNA. The remaining downstream 30 RNA trailing out of the transcribing Pol II is attacked by a 50 -30 exonuclease. The nuclease rapidly degrades the RNA from its free 50 end and, when it eventually catches up to the elongation complex, somehow destabilizes the EC and causes its termination. Some precursor-mRNAs, such as human b-globin premRNA, possess two cleavage sites, the normal polyadenylation site and a further downstream, the cotranscriptional cleavage (CoTC), site. Interestingly, it was found that the RNA sequence at the CoTC forms an RNA enzyme (a ribozyme) with a structure that has intrinsic self-cleavage activity in the absence of proteins (Teixeira et al, 2004). Cotranscriptional cleavage in the CoTC site of the nascent b-globin pre-mRNA is required for efficient termination (West, et al., 2004). Termination also depends on the presence of the poly(A) site in the nascent b-globin premRNA (Dye and Proudfoot, 1999; West et al., 2004). Knockdown and depletion of the 50 -30 exonuclease, or mutational destruction of the CoTC site, strongly stabilized the RNA fragment downstream from the poly(A) cleavage site, indicating that the exonuclease is responsible for degrading this region of the nascent transcript (Kim et al., 2004b; Teixeira et al., 2004; West et al., 2004). Furthermore, Pol II could be chemically crosslinked to sequences in the encoding gene that were farther downstream from the poly(A) site in the cells in which the 50 exonuclease was depleted than in the normal cells, suggesting that without functional 50 -30 exonuclease, Pol II fails to stop when it should (Kim et al., 2004b; West et al., 2004). Hence, one interpretation of the mechanism of transcription termination is, of ‘‘a race between the 50 -30 exonuclease and the elongating RNA polymerase, and cleavage of the nascent mRNA at the CoTC lets the nuclease jump ahead by providing a downstream entry site, making the termination more efficient’’ (Tollervey, 2004), since cleavage at the poly(A) site is a relatively slow process. Apparently, there are certain distinctions between the Pol II transcription termination in higher eukaryotes and lower eukaryotes. Genes of lower eukaryotes (such as yeast) have only one transcript cleavage site, the poly(A) site, and the 50 -30 exonuclease called Rat1 requires at least one cofactor called Rai1, and perhaps a second one called Rtt103 (Kim et al., 2004b). By contrast, the human nascent b-globin pre-mRNA has two cleavage sites, the normal poly(A) site and the downstream, auto-cleaving CoTC site (Teixeira et al., 2004). The 50 end generated by the CoTC cleavage and not the poly(A) site is the actual substrate for degradation by a 50 -30 exonuclease (Xrn2) (West et al., 2004). Previously, 50 -30 exonucleases such as Xrn2 have been shown to require a 50 phosphate for activity (Stevens and Poole, 1995). A 30 -50 exonuclease activity as part of a multi-subunit complex (exosome) may quickly degrade the nascent transcript back to the poly(A) signal (Torchet et al., 2002; West et al., 2004).

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The above studies clearly implicated the 50 -30 exonuclease in transcriptional termination by the ‘‘torpedo’’ model. However, several observations indicate that this may not be the complete mechanism for Pol II termination of transcription: (1) Since transcriptional termination is required by every transcribed gene, it is reasonable to expect, in addition to common RNA processing and termination factors, presence of conserved nucleotide sequences at and around the main cleavage site (the poly(A) site) for all genes of an organism, and to a large extent between genes of different organisms. Curiously, no such conserved sequences have been found. This could suggest the possible involvement of a ‘‘gene-specific’’ factor that binds to this variable region of a gene and is required for the specific cleavage of the nascent transcript. Appropriate mutational studies of this sequence-variable region, between the poly(A) site and the DSE, could illuminate the relevance of these sequences to the transcriptional termination. (2) From the perspective of a cell, it seems a lot simpler and more efficient if the poly(A) site were cleaved by a simple endonuclease, or auto-catalytically (by a ribozyme), similar to the CoTC site, followed by polyadenylation. Instead, a very complex process for cleavage of the poly(A) site has developed and conserved for eukaryotes that involves participation of many factors as listed above. Conservation of such a complex and possibly gene-specific process may be indicative of coupling of transcription termination to other important cellular processes. (3) Depletion of the 50 exonuclease is expected to cause serious transcriptional termination defects in every protein-encoding gene of a cell that collectively would have lethal effect on the cell. Moreover, depletion of the 50 exonuclease is expected to yield a roughly uniform Pol II density throughout the termination region extending to hundreds of nucleotides beyond its poly(A) site. The results of the above referenced studies provide no clear indication of either an absolute viability problem for the cell or a uniform Pol II density under the condition of the 50 exonuclease depletion. In the 50 exonucleasedepleted cells compared to normal cells, Pol II was observed much farther downstream of the poly(A) site, declining at a more gradual rate, and the transcription in the termination region was markedly extended (Kim et al., 2004b; West et al., 2004). However, the data indicate that termination did eventually occur, albeit ineffici ently and with certain delay. Such a profile could suggest the existence of a more complex mechanism for Pol II transcription termination. (4) In the human nascent b-globin pre-mRNA, presence of the poly(A) site is required for proper transcriptional termination, even though the 50 end generated by the poly(A) site cleavage is not a substrate for the 50 -30 exonuclease (West et al., 2004). This is a further indication that the 50 -30 exonuclease activity is not the complete mechanism for Pol II transcription termination. Further evidence for this notion comes from the studies on the allosteric model (see below).

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3. Allosteric model Considerable evidence suggests that the CTD of Pol II functions in the cotranscriptional recruitment and/or assembly of protein factors involved in cleavage-polyadenylation (for reviews, see Hirose and Manley, 2000; Proudfoot and O’Sullivan, 2002; Zorio and Bentley, 2004). It is plausible that some of the cleavage-polyadenylation factors that are recruited by CTD also mediate termination through this interaction in response to transcription of the poly(A) signal. In this regard, yeast or Drosophila Pcf11 (Zhang et al., 2005; Zhang and Gilmour, 2006, respectively), which associates directly with RNA and the CTD via its CTD-interacting domain (CID), is capable of dismantling an in vitro assembled EC. Since cleavage of the pre-mRNA is a prerequisite for the torpedo model, support for the allosteric model has come partly from the studies showing that termination can occur in the absence of the prior cleavage. Electron microscopy (EM)-visualization of plasmid-driven transcription in Xenopus oocyte nuclei revealed that although there is a correlation between poly(A) signal strength and termination efficiency, prior cleavage of the nascent transcript is not a prerequisite for termination (Osheim et al., 1999). Further EM survey of more than 100 Pol II-transcribed genes in Drosophila showed that whereas cotranscriptional cleavage did precede termination in some genes, in the majority the full-length transcript was released from the template before the cleavage event (Osheim et al., 2002). Observation that mutations in the complex poly(A) signal of yeast FBP1 gene that strongly impair cleavage and polyadenylation have little effect on termination efficiency led to the hypothesis that the assembly and recruitment of a partial cleavage-polyadenylation complex may be sufficient for termination (Aranda et al., 1998). Supporting the idea that cleavage-polyadenylation is not a prerequisite for termination are the transcription run-on experiments demonstrating that these activities are separable on different domains of some factors involved in both cleavage-polyadenylation and termination, but not in the 50 -30 exonuclease activity (Sadowski et al, 2003; He et al., 2003). But, recent data (Luo et al., 2006) show that the cleavage and termination functions of these factors may be inseparable (see Section 6). Other factors with positive role in changing the conformation of EC and promoting termination include the G1-S transition-specific factor MBF components Mbp1 and Grs1 (Aranda and Proudfoot, 2001; Magrath and Hyman, 1999). While these experiments do not exclude a role for cleavage in efficient termination, they are consistent with the notion that a conformation change of the EC is required, and in some cases might be sufficient, for termination. Additional support for the allosteric model comes from analysis of proteins that have no effect on cleavage or polyadenylation, yet their loss or recruitment might affect the speculative conformational change of the EC that is central to this model. For example, Sub1, yeast homolog of

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the mammalian transcriptional coactivator PC4, inhibits premature termination by interacting with Rna15. Cells having a mutation in Rna15 that increase its affinity for Sub1 show termination defects that could be suppressed by deletion of SUB1 (Calvo and Manley, 2001). Similarly, anti-terminator activity was shown for the mRNA-binding protein NPl3 (Bucheli and Buratowski, 2005). Release of Sub1 and MPl3 following transcription of the poly(A) signal might result in a conformational change in the EC, leading to termination. Despite influencing termination, it is not known whether the transcription of the poly(A) signal affects the association of various factors with the EC, or their activity, nor is it known through what mechanism they influence termination (Rosonina et al., 2006). 4. GENEi model Short double-stranded RNA (dsRNA) is a natural regulator of gene expression in eukaryotes triggering various gene-silencing mechanisms that are collectively referred to as gene silencing or RNA interference (RNAi). Sequence-specific mRNA degradation is induced by 21–23nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III (Dicer) cleavage from longer dsRNA (Meister and Tuschl, 2004). Bahramian and Zarbl (2005a) proposed a model where siRNAs are involved with the accelerated decay and inhibition of translation of stable mRNA observed following transcription shutdown. This model, called GENE impedance (GENEi), unifies several biological phenomena, including the metabolism of stable mRNA, RNAi, transcription-termination, transcriptionarrest, endogenous synthesis of anti-sense RNA and perhaps micro-RNA, and cellular defense mechanisms invoked by invading genetic elements. GENEi model should generate considerable interest among those investigators engaged in mechanistic analysis of transcription, particularly its role in transcription termination and siRNA-mediated inhibition of gene expression. The basis for Bahramian and Zarbl (2005a)’s theoretical model is that gene expression is discontinuous and responsive to cellular demand for a particular gene product at a given time. Based on our published results on the transcriptional and post-transcriptional silencing of rodent pro-a1(I) collagen gene by homologous transgenes and the literature on siRNA, we have postulated that there is a cellular mechanism to coordinate efficient and specific transcriptional inhibition, degradation of the mRNA and inhibition of translation of the transcripts of a target gene in response to physiological signals. We discovered that not only were certain transiently transfected procollagen genes totally silent soon after transfection, but their presence in the cell nucleus also caused a rapid and dramatic reduction in the steady-state level of the endogenous stable procollagen transcripts. Transient transfection with, for example, a construct that carried both the 50 regulatory regions and the 30 end sequences of the gene, resulted in rapid decline in

the steady-state levels of the endogenous gene transcripts by greater than 90% in non-transformed and v-fostransformed mammalian cells. Following this transfection, the endogenous procollagen transcripts were degraded at a highly accelerated rate. Also, the effect on mRNA stability persisted for days after transfection. So we inferred the existence of an active process of specific RNA degradation. We postulated that the stable mRNA decay represents the post-transcriptional component of a fundamental process for impedance of gene expression, one that comprises the dsRNA-dependent short interfering RNA (siRNA) mechanism. According to the GENEi hypothesis, transcription repression essentially utilizes the machinery and components normally used in transcription elongation, processing and termination. Additionally, GENEi proposes that genespecific sequence elements and predicted cognate factors called ‘‘co-activator(s)’’ and ‘‘transcription repression unit’’ (TRU) contribute to the control of gene expression. Indirect evidence was presented that the co-activators binding to specific 50 regions of the procollagen gene regulate initiation and elongation of transcription, while binding of TRU to the 30 region of the gene regulates transcription termination and arrest. According to the model, each round of transcription by Pol II is terminated by interaction of the polymerase with a transcription termination/repression complex (TTRC) located in the termination region of a gene, close to the poly(A) cleavage site. It has been proposed that the TTRC consists of two cooperative components, the gene-specific TRU that is essential in cleavage of the transcript at the poly(A) site, thus separating the nascent RNA from Pol II, and a general transcription termination unit (TTU). The main function of TTU is to facilitate release of the Pol II from the DNA template at the termination region of a gene. Additionally, it could cooperate with TRU in cleavage of the nascent transcript. When TRU is allosterically altered (intrinsic GENEi) or depleted (alternative GENEi) during transcription shutdown, cleavage of the nascent transcript cannot occur; but the TTU-mediated release of the Pol II from the DNA template proceeds and is somehow coupled to the synthesis of anti-sense RNA. The interaction of the Pol II with TTU, therefore, may result in a conformational change in the EC that not only impedes its progression on the sense DNA strand, but somehow promotes synthesis of anti-sense RNA from the 30 end that results in formation of dsRNA, either in the form of a hairpin or sense-anti-sense RNA. The dsRNA is subsequently cleaved to duplexes of 22 nucleotides in length by Dicer, which are directed to specific cleavage and inhibition of translation of the cognate mRNA using the RNAi pathway. The GENEi model is supported by the finding that generation of the endogenous siRNA begins with the synthesis of longer precursors of dsRNA from the 30 region of a gene undergoing repression. The presence of human RISCs (siRNA- and micro-RNA-programmed RNA-induced silencing complexes) in the nucleus and knock down

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of target RNA levels (Robb et al., 2005) corroborates Bahramian and Zarbl’s hypothesis that assembly and functionality of RNAi machinery could begin in the nucleus then extend to the cytoplasm. While linking the in vivo production of siRNA to the degradation of stable mRNA was an important discovery, some features of the GENEi model remained unsolved. The exact positions and sequences of the cis-acting elements, as well as the molecular identities of the relevant binding factors (i.e. co-activator(s) and TRU) and the TTU are unknown. Above all, the exact mechanism of dsRNA synthesis, which must take place during transcription shutdown, is unknown. Previously we hypothesized that the interaction between TRU and TTU is the key determinant for the Pol II either to proceed with termination of transcription, or to engage in dsRNA synthesis that leads to RNA interference. The interaction between TRU and TTU may not be necessary, therefore, requiring a modification to our model. TTU could be a component of the cleavage-polyadenylation and termination complex that is recruited by the transcribing Pol II during termination of transcription (see below).

5. Integrated allostric-GENEi-torpedo model The GENEi model couples the process of termination of transcription by Pol II to the synthesis of anti-sense RNA originating in the termination region of a gene, which under certain condition could extend to the sequences upstream of the poly(A) site, thus generating dsRNA. Conformation of a gene-specific factor (TRU) influenced by the expression status of a gene, either in repressed or active transcription mode, could determine, respectively, whether synthesis of the anti-sense RNA should continue beyond the poly(A) cleavage site or not. Synthesis of the anti-sense RNA would reasonably require some form of RNA-dependent RNA polymerase (RdRP) activity, as a hundred and eighty degree directional rotation of Pol II complex to switch from the template DNA strand to the complementary DNA strand seems mechanistically impossible (Bahramian and Zarbl, 2005b). Some RdRP could participate in de novo synthesis of RNA complementary to the nascent transcript. RdRPs, which are believed to play an important role in RNAi process and are required for a variety of cellular functions, have been identified in several organisms including plants (Dalmay et al., 2000; Schiebel et al., 1993), fungi (Cogoni and Macino, 1999; Martens et al., 2002; Volpe et al., 2002), and Caenorhabditis elegans (Sijen et al., 2001; Smardon et al., 2000). However, no RdRP homologs have been identified in Drosophila or mammals, leading to the suggestion that RNAi can occur in the absence of RdRP activity (Sugiyama et al., 2005). Conversion of the transcribing Pol II to RdRP by a cofactor, and using the nascent RNA as template for synthesis of anti-sense RNA, is a possibility (Bahramian and Zarbl, 2005b).

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The TTU could be visualized as a ‘‘converter’’ factor that upon interaction with transcribing Pol II allosterically alters and converts it to an RNA-dependent RNA polymerase. It is proposed that TTU, as a component of cleavage-polyadenylation and termination complex, is loaded onto the CTD of Pol II near the poly(A) cleavage site (Figs. 1A and 2A), or other pause sites present in the termination region. The TTU-induced conformational change to the Pol II alters its template preference from DNA to RNA. The switch from the DNA template to the nascent RNA, which is still attached by its 30 end to the active site of the enzyme, could be a slow process. Therefore, Pol II transcription on the DNA template could continue for several hundred nucleotides beyond the poly(A) site before switching onto the RNA template occurs. This would allow time for also a slow cleavage reaction at the poly(A) site to happen. Formation of secondary structures on the nascent RNA could facilitate the template switching by bringing the nascent RNA in correct orientation to the proximity of DNA template at the active site of the enzyme (see Figs. 1A and 2A). The switching of template by Pol II from DNA onto RNA marks the end of DNA-dependent RNA polymerase activity and the beginning of RNA-dependent RNA polymerase activity that reads the template RNA from 30 to 50 direction and adds complementary nucleotides to the 30 end, resulting in the formation of dsRNA. This transcription could continue up to the poly(A) cleavage site if RNA cleavage and polyadenylation had occurred. In the mean time, the 50 exonuclease (Rat1, in yeast), assisted by its cofactors (Rai1 and Rtt103), having off-loaded onto the 50 end of the downstream RNA, which has become the template RNA, degrades the downstream RNA while proceeding toward the polymerase (Fig. 1B). Rat1 destabilizes the polymerase by destroying both its template and its product, thus resulting in proper termination (Fig. 1C). In the absence of a functional TRU, as a consequence of its interaction with a repressor (‘‘intrinsic GENEi’’) or removal by competing transgenes (‘‘alternative GENEi’’) (Bahramian and Zarbl, 2005a), the pre-mRNA is not cleaved at the poly(A) site and the 50 -30 exonuclease is not loaded, but switching of template from DNA to RNA promoted by the TTU converter occurs. In this case, synthesis of the anti-sense RNA continues past the poly(A) site, yielding dsRNA homologous to the mRNA sequence (Fig. 1D), which would be cleaved by Dicer that leads to RNAi. In higher eukaryotic genes, such as human b-globin gene, presence of a CoTC site several hundred nucleotides downstream of the poly(A) site causes auto-cleavage of the nascent RNA at this site. This cleavage provides an entry site for the 50 exonuclease (Xrn2) onto the free 50 end of the 30 RNA (Fig. 2B). Digestion of the 30 RNA by Xrn2 destabilizes the polymerase complex and terminates transcription (Fig. 2C). The RNA fragment immediately downstream of the poly(A) site (poly(A)—CoTC), whether the poly(A) site is cleaved or not, is digested by a

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single-strand-RNA 30 ribonuclease, as this RNA is not a substrate for Xrn2 (Fig. 2C). In the absence of cleavage at the poly(A) site (GENEi), synthesis of anti-sense and generation of dsRNA extends to the region upstream of the poly(A) site (Fig. 2D), provided that CoTC occurs not before the polymerase switches template and retraces its path back along the nascent RNA and passed the CoTC signal. The resulting dsRNA is digested by Dicer to fragments of 22 bp that initiate RNAi. 6. Discussion The topic of nucleotide sequence specific doublestranded RNA (dsRNA)-mediated gene silencing is of great interest. Previously, we have established that at least the post-transcriptional feature of GENEi operates via synthesis of dsRNA that originates in the termination region of a gene and comprises sequences homologous to a coding segment of the gene (Bahramian and Zarbl, 2005a). However, the exact mechanism by which the dsRNA is synthesized remained unidentified. This paper describes a plausible mechanism by which the endogenous dsRNA could be generated, which appears to be linked to the mechanism of transcription termination by Pol II. In the cytosol we detect mRNAs each with a polyA tail at its 30 end. When transcribing such mRNAs from a DNA template, RNA polymerase, contrary to logic, does not just stop at the point where the polyA tail is added to the mRNA, but continues transcribing until some point when it departs from the template. Thus, the pre-mRNA is longer than the mRNA, and has to be cleaved to generate the site for 30 addition of the polyA tail. The apparently superfluous 30 extension is degraded by appropriate ribonucleases. In this paper, Bahramian suggests that the 30 extension is not superfluous. It gives an opportunity for the examination of options, other than being degraded. In particular, it is proposed, according to the preferred allosteric-GENEitorpedo model, that the DNA-dependent RNA polymerase acquires RNA-dependent RNA polymerase activity and retraces its path, back along the RNA that has just been synthesized, so generating dsRNA. The extent to which it can retrace this path is determined by the factors influencing the cleavage of the pre-mRNA at the site of polyA addition. If cleavage occurs, the retracing is cut short. If the cleavage is inhibited then a full-length dsRNA can be produced. This can then be subject to cleavage by ‘‘Dicer’’ that leads to the induction of RNAi. Indirect support for the GENEi model and, therefore, the integrated allosteric-GENEi-torpedo model, is provided by the investigations of the role of intergenic transcription in gene and chromatin activation (Haussecker and Proudfoot, 2005). The widespread occurrence of intergenic transcription in eukaryotes has led to the proposal of models that implicate a role for such transcription in gene and chromatin activation. Analysis of intergenic transcription and the chromatin state

throughout the human b-globin gene cluster found the data not to be consistent with such activation-linked models; the intergenic transcription levels neither correlated with chromatin activation nor with globin gene expression (Haussecker and Proudfoot, 2005). Strikingly, both sense and anti-sense intergenic transcripts were readily detectable. In the light of mounting evidence that RNAi plays in the epigenetic organization of genomes from a diverse range of organisms, including vertebrates, these authors, therefore, investigated the involvement of RNAi-related processes in regulating intergenic transcript levels in the b-globin gene cluster. If Dicer were involved in turning over b-globin intergenic transcripts, then reduced Dicer activity would be predicted to lead to an increase in intergenic transcript levels. This was indeed the case. Both sense and anti-sense intergenic b-globin transcripts were specifically upregulated in cells knocked down for Dicer. Similar to intergenic transcripts, unspliced globin transcripts were strongly upregulated in Dicer-knockdown cells, whereas spliced globin transcripts were much less affected. Both the unspliced globin transcripts and the intergenic transcript are only present in the nucleus. The results suggested that the nuclear RNAi-related processes are closely coupled to transcription [and presumably transcriptional termination]. Further investigations of the mechanisms of dsRNA synthesis in the intergenic and genic regions of a gene could illuminate the possible relevance of this process to transcription termination as well as gene silencing. Several DNA-dependent RNA polymerases have been shown to possess the intrinsic ability to use RNA molecules as templates when a suitable one is offered. Small RNA viruses such as the human hepatitis delta virus (Taylor, 2003) and plant viroids (Flores et al., 2004) are known to be able to divert RNA polymerase II for the synthesis of their own genomic RNA without DNA intermediates. Thus, it is possible that Pol II may switch to the nascent RNA in the termination region of an endogenous gene, assisted by the TTU ‘‘converter’’ factor. It may seem difficult for a complex enzyme such as Pol II to switch template and at the same time change course through 1801, but this could be facilitated by the formation of hairpin structures on the nascent transcript. Moreover, such acrobatics by Pol II is not totally unfamiliar, since, for example, a physical association of promoter and termination regions of yeast BUD3 and SEN1 genes have been reported that are transcription-dependent, require the Ssu72 and Pta1 components of the CPF 30 -end processing complex, and require the phosphatase activity of the Ssu72 (Ansari and Hampsey, 2005). On the basis of these results, the authors have proposed a model for Pol II transcription in which promoter and terminator regions are juxtaposed, and that the resulting gene loops facilitate transcription reinitiation by the same molecule of Pol II in a manner dependent on Ssu72-mediated CTD dephosphorylation. Further investigations of DNA to RNA template switching by Pol II may elucidate the mechanism of transcription

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termination by this complex enzyme, as well as dsRNAinduced and transgene-induced gene silencing, therefore, validate the allosteric-GENEi-torpedo model, which may be considered as a sophisticated version of the allosteric/ torpedo model. Strong support for the torpedo model was provided by the above studies on the Rat1 and Xrn2 50 -30 exonucleases showing that they are required in catalytically active form for termination. Paradoxically, other data discussed above suggested that cleavage of the nascent RNA is not required for transcription termination. However, cotranscriptional degradation of the nascent RNA was not directly demonstrated. Recent studies have shown that the two 50 -30 exonucleases of yeast, Rat1 and Xrn1, both contribute to cotranscriptional degradation of nascent RNA, but this degradation is not sufficient to cause polymerase release (Luo et al., 2006). Unexpectedly, they found that Rat1 functions in both 30 -end processing and termination by enhancing recruitment of 30 end processing factors, including Pcf11 and Rna15. Additionally, the cleavage factor Pcf11 reciprocally aids in recruitment of Rat1 to the elongation complex. On the basis of these results, they suggested a unified allosteric/torpedo model in which Rat1 is not a dedicated termination factor, but is an integrated component of the cleavage/polyadenylation apparatus. The integrated ‘‘allosteric-GENEi-torpedo’’ model requires participation of at least two putative pre-mRNA processing and termination factors, the gene-specific and allosteric factor TRU, and the Pol II-to-RdRP converter factor TTU. These factors eventually could be identified with some factors that are already purified, or could turn out to be novel factors. The TRU is less likely to have been isolated, as is predicted to be DNA-associated while participating in the poly(A) site cleavage. One known factor that could fulfill the role of TTU is Pcf11. Pcf11is a termination factor that is concentrated at the 30 end of a gene, and is thought via its CID to be able to dismantle the EC by bridging the CTD of Pol II to the nascent transcript (Zhang et al., 2005). Depletion of Drosophila Pcf11 with RNAi causes Pol II to readthrough the normal region of termination (Zhang and Gilmour, 2006), suggesting that it might play a central role in template release. Initial genetic evidence pointed to separable functions of Pcf11 in 30 -end processing and in termination, indicating that normal termination was detected in mutants with defects in cleavage and polyadenylation (Sadowski et al., 2003). Recent evidence (Luo et al., 2006), however, shows that the same Pcf11 mutation previously shown to affect only cleavage in fact causes both cleavage and termination defects. Therefore, it is unclear whether Pcf11 can function in termination independent of its activity as mRNA 30 -end processing factor. In an attempt to explain the discrepancy between their result and that of Sadowski et al. (2003), Luo et al. (2006) stated that unlike chromatin immunoprecipitation (ChIP) assay used by them, the run-on assay used in the previous work would not detect polymerases that failed

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to terminate if they did not resume elongation under the in vitro conditions used. Other factors, in addition to Pcf11, could be responsible for dissociating Pol II ECs. Considering the evidence that termination factors, Rho in bacteria (Nudler and Gottesman, 2002), NPH-I in vaccinia virus (Deng and Shuman, 1998), and Sen1 in yeast snRNA and snoRNA genes (Steinmetz et al., 2001) use ATP hydrolysis and likely act as helicases to disrupt RNA–DNA hybrids within the RNA polymerase active site, Rosonina et al. (2006) suggested that a similar factor is required to dissociate Pol II from the template DNA of mRNA-encoding genes. Transcription termination factor 2 (TTF2) is a well-characterized Pol II-associated factor, with DNA-dependent ATPase activity and a helicase domain (Jiang et al., 2004), that may fit the bill (Rosonina et al., 2006). This could work in association with Pcf11 and/or another nascent RNA-binding factor(s), such as Rat1/Xrn2, or Rna15. Such cooperative termination activity is well documented in bacteria and vaccinia virus RNA polymerase (Deng and Shuman, 1998; Nudler and Gottesman, 2002). There is also some evidence for the notion of repressor molecules interacting with gene-specific factors at the 50 and the 30 ends of a gene: In the case of type I procollagen, amino- and carboxyl-terminal propeptides have been shown to participate in pre-translational regulation of the collagen synthesis (Wu et al., 1986). All collagens are initially produced as precursors procollagens, which are larger than the final product by virtue of extension propeptides at the amino and carboxyl termini of the molecule. During the conversion of procollagen to collagen, the propeptides are cleaved by specific proteases (Leung et al., 1979). Using amino-terminal propeptides in vitro, various investigators have demonstrated feedback inhibition of collagen synthesis (Horlein et al., 1981; Paglia et al., 1979). To assess possible pre-translational effects of propeptides, human lung fibroblasts (IMR-90) were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a cDNA probe (Wu et al., 1986). Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative non-collagenous protein, b-actin, were unaffected by either propeptide throughout the concentration ranges. In eukaryotes, polyadenylation of mRNA protects transcripts from degradation and enhances translation efficiency. Conversely, in bacteria, polyadenylation destabilizes mRNA. Recent studies have implicated RNA adenylation in promoting degradation of some RNAs in eukaryotes by exosome. The exosome is a nuclear complex of proteins with exonuclease activity that is conserved in

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eukaryotes. The human b-globin pre-mRNA and mouse serum albumin pre-mRNAs are cotranscriptionally cleaved within their 30 flanks. CoTC produces two naked RNA ends, both subject to rapid degradation (West et al., 2006). Degradation of the 30 product by the 50 -30 exonuclease Xrn2 is associated with transcriptional termination (West et al., 2004). The 50 products of CoTC accumulate upon knockdown of the exosome subunits, and some of them carry short A tails. The stabilizing role of mRNA 30 end polyadenylation is brought about by binding of nuclear poly(A)-binding protein (PABPN1) to the poly(A) tail, blocking degradation and increasing polyadenylation efficiency. A minimum of ten adenines are needed for PABPN1 binding. Thus, short A tails, such as in CoTC products, will not bind PABPN1 and are vulnerable to 30 exonucleolytic attack. A single G residue incorporated into three of the sequenced A tails is a higher frequency than would be expected for poly(A) polymerase (PAP), which led to the anticipation that an enzyme other than PAP might be responsible for adenylating CoTC ends prior to exosome degradation (West et al., 2006). Addition of short A tails to the CoTC products has no conflict with the proposed integrated ‘‘allosteric-GENEi-torpedo’’ model. The integrated allosteric-GENEi-torpedo model offers mechanistic solutions to the problems of transcription termination, RNAi and gene silencing phenomena. Particularly, the notion of Pol II EC acquiring a new function of RNA-dependent RNA polymerase activity through its interaction with the putative TTU during termination is an interesting one. This could also work in conjunction with other probably required activities, such as ATP-dependent helicase and transcription initiation factors. The anticipated requirement of a gene-specific, allosteric factor TRU in offering a choice between transcription termination and gene silencing is also an interesting hypothesis that deserves investigation. Future studies could lead to the illumination of these phenomena and support for the proposed integrated model that couples termination of transcription to RNA interference and gene silencing. Acknowledgments I am grateful to Dr. D.R. Forsdyke (Canada) for constructive review of this paper and helpful suggestions. I thank Drs. I. Altosaar (Canada) and H. Zarbl (USA) for reading the initial draft of the manuscript and comments. Competing interests statement: No competing financial interests declared. References Ahn, S.H., Kim, M., Buratowski, S., 2004. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 30 end processing. Mol. Cell 13, 67–76. Ansari, A., Hampsey, M., 2005. A role for the CPF 30 end processing machinery in RNAP II-dependent gene looping. Genes Dev. 19, 2969–2978.

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