Transcription termination by nuclear RNA polymerases

Rat1/Xrn2: the transcription termination torpedo

The torpedo model is strongly supported by studies that demonstrated a role for the yeast 5′–3′ exonuclease Rat1 and its human homolog, Xrn2, in RNAPII termination (Kim et al. 2004b; West et al. 2004). In both cases, inactivation of the exonuclease resulted in defects in termination, as measured by ChIP and/or nuclear run-on experiments, both methods measuring RNAPII density throughout transcribed genes. Rat1 and its cofactor Rai1 (see Xiang et al. 2009) are strongly associated with the 3′-end of genes, although in a manner independent of Ser2 CTD phosphorylation since a deletion of CTK1 does not affect their association (Kim et al. 2004b). Recruitment of Rat1 is more likely indirect, through 3′-end processing factors. Rtt103 is a Ser2 phosphorylated CTD-binding protein that cross-links to the 3′-ends of genes in ChIP experiments, and copurifies with Rat1 and other subunits of the cleavage/polyadenylation complex (Kim et al. 2004b). The Caenorhabditis elegans homolog of Rtt103, CIDS-1, was recently identified in a genome-wide RNAi screen for factors involved in termination, and deletion of cids-1 resulted in reduced 3′-end cleavage, supporting the connection between cleavage and termination (Cui et al. 2008). Additionally, Rat1 recruitment to the 3′-end of a protein-coding gene was reduced by Pcf11 inactivation. Similarly Rat1 is required for the recruitment of Pcf11 and Rna15, the yeast CstF-64 equivalent, to the 3′-end of genes (Luo et al. 2006). These data suggest that Rat1 is important for the efficient recruitment of 3′-end processing factors and/or vice versa. However, depletion of Rat1 does not affect cleavage at the poly(A) site (Kim et al. 2004b).

Rat1 activity is likely not sufficient to bring about RNA and RNAPII release from the template. Depletion of Rat1 inhibits but does not abolish termination (Kim et al. 2004b), and digestion of RNAPII-associated RNA is not sufficient to disassemble the EC in vitro (Gu et al. 1996; Kireeva et al. 2000). Consistent with this, the abundant and mostly cytoplasmic exonuclease Xrn1, which is similar to Rat1, is able to complement the exonuclease activity of a Rat1-deficient strain when targeted to the nucleus, but unable to complement the termination defect (Luo et al. 2006). Moreover, a defect in termination in a RAT1 mutant strain (Kim et al. 2006) is not associated with stabilization of the 3′ downstream product, providing evidence that degradation of the nascent 3′ product is not sufficient to promote termination (Luo et al. 2006).

The above results suggest that another activity, perhaps associated with Rat1, is required to efficiently dismantle the EC. Indeed, Kawauchi et al. (2008) provided evidence that Sen1, an RNA/DNA-dependant ATPase and a member of the superfamily I of RNA/DNA helicases (Kim et al. 1999), cooperates with Rat1 to promote efficient RNAPII termination at two protein-coding genes. They suggested that Sen1 might expose the downstream RNA product generated after cleavage to facilitate its degradation by Rat1. Another possibility is that it could function at the site of termination to help disrupt the ternary complex. Furthermore, even though Sen1 was previously detected at snoRNAs genes (Kim et al. 2006), read-through at noncoding and some coding genes in a SEN1 mutant strain supports Sen1 involvement in termination of both class of RNAs (Steinmetz et al. 2006b). Sen1 is a large, low-abundance nuclear protein that interacts with the CTD (Ursic et al. 2004) and is implicated in processing of a variety of noncoding RNAs (Winey and Culbertson 1988; Ursic et al. 1995).

Senataxin (SETX) is the putative human homolog of Sen1 and possesses a conserved helicase domain (Moreira et al. 2004). Interestingly, mutations in SETX lead to two neurological disorders, an autosomal dominant form, ALS4 (amyotrophic lateral sclerosis type 4) (Blair et al. 2000), and an autosomal recessive form, AOA2 (ataxia-oculomotor apraxia type 2) (Moreira et al. 2004; Chen et al. 2006). Although the only characterized role of SETX in humans is its involvement in DNA damage repair (Suraweera et al. 2007), it will be interesting to determine whether SETX also functions in transcription termination, particularly in neurons.

The Rat1 homolog in humans is Xrn2, and its function in termination appears to be conserved. West et al. (2004), using siRNA-mediated knockdown, provided evidence that Xrn2 degrades the downstream 3′ RNA of the β-globin transcript and promotes termination. However, the effect of Xrn2 on β-globin pre-mRNA termination is specific to a cotranscriptional cleavage (CoTC) activity and not to the poly(A) site. Indeed, the β-globin gene contains a specific CoTC element, ∼1 kb downstream from the poly(A) site, that is required for termination (Dye and Proudfoot 1999, 2001). The CoTC sequence undergoes a rapid cleavage event that exposes a free 5′ monophosphorylated downstream RNA that serves as a substrate for Xrn2 (Teixeira et al. 2004; West et al. 2004). It is important to note that CoTC appears to be a specific termination signal for some globin genes (it is also found in the ε-globin 3′ flanking region) (Dye and Proudfoot 2001) that has not yet been identified in other genes. In most genes, which lack CoTC, cleavage at the poly(A) site generates the entry site for Xrn2.

Like Rat1, Xrn2 interacts with processing factors that might facilitate its recruitment to the 3′-ends of genes. Kaneko et al. (2007) showed that Xrn2 interacts with CstF-64 and p54nrb/PSF (protein-associated splicing factor). p54nrb and PSF are related multifunctional proteins that are involved in transcription, splicing, and polyadenylation (Rosonina et al. 2005; Liang and Lutz 2006). ChIP experiments revealed that, while p54nrb/PSF, cross-linked along the length of the β-actin gene, Xrn2 cross-linked primarily downstream from the poly(A) site, a result consistent with Rat1 localization along yeast genes (Kim et al. 2004b; Luo et al. 2006). In vitro cleavage assays demonstrated that while Xrn2 and p54nrb/PSF are required for degradation of the 3′ cleavage product, they are not necessary for cleavage itself (Kaneko et al. 2007). Coupling of Xrn2 to 3′ processing, however, was shown to stimulate exonuclease activity in the nuclear extract. These experiments, together with the observation that knockdown of p54nrb interferes with Xrn2 recruitment and termination, suggest that Xrn2 is recruited to the cleavage/polyadenylation machinery via p54nrb/PSF. The involvement of p54nrb/PSF in termination was recently confirmed in a genome-wide RNAi screen in C. elegans (Cui et al. 2008).

Evidence supporting the hybrid model of termination came from recent experiments that separated pre-mRNA associated with the DNA template from the released RNA. West et al. (2008) provided evidence that poly(A) site cleavage can occur while RNAPII is still bound to the template, and that Xrn2-mediated degradation of the RNAPII-associated downstream RNA precedes RNAPII release from the template. Alternatively, cleavage at the CoTC site in the terminator region of the β-globin gene allows Xrn2 entry and leads to RNAPII release before cleavage at the poly(A) site. However, in both cases, poly(A) site recognition, which occurs rapidly, is essential for termination. These data reinforce the idea that transcription termination is a combination of both the allosteric and torpedo models (Fig. 1).

Pausing the polymerase: another factor in RNAPII termination

ChIP analysis of human genes frequently shows a high density of RNAPII at promoter regions that decreases throughout the gene body and increases downstream from the poly(A) site (Glover-Cutter et al. 2008). This increased RNAPII density at the 3′-end may reflect pausing of RNAPII that precedes termination. Indeed, transcription pause sites located downstream from poly(A) sites appear to play a role in RNAPII 3′-end processing and termination, at least in mammalian genes, such as α-globin and γ-globin genes (Enriquez-Harris et al. 1991; Plant et al. 2005). A G-rich sequence (MAZ element: G₅AG₅) was identified downstream from the poly(A) site of the liver-specific C2 complement gene (Ashfield et al. 1994; Yonaha and Proudfoot 1999, 2000). This sequence is a potential binding site for the transcription factor MAZ and promotes poly(A)-dependant termination. Indeed, siRNA-mediated depletion of Xrn2 decreased MAZ-dependent termination efficiency (Gromak et al. 2006). Additionally, Gromak et al. (2006) described another G-rich region downstream from the β-actin poly(A) site that acts as a pause site-mediated terminator. Interestingly, the strength of the poly(A) site correlates with efficient pausing-dependent termination (Plant et al. 2005; Gromak et al. 2006).

Other cis-acting protein-binding sites have been shown to function in termination. It has been suggested that the poly(A) signal itself can direct RNAPII pausing. The AAUAAA-dependent pausing is mediated by CPSF but does not require the CTD and any other downstream pausing elements (Orozco et al. 2002; Park et al. 2004; Nag et al. 2006, 2007). Another sequence, a CCAAT-box in the adenovirus late promoter is necessary for termination of the upstream gene (Connelly and Manley 1989). West et al. (2006b) showed that multiple termination sequence elements in the mouse serum albumin (MSA) 3′-flanking region promote termination. Many MSA transcripts appear to be cleaved cotranscriptionally and, interestingly, seem to be adenylated and processed by the TRAMP/exosome complex (West et al. 2006a,b). Whether these sequence elements are implicated in RNAPII pausing remains to be elucidated. Pausing also plays a role in RNAPI and RNAPIII termination (see below), making it a general feature of the termination process. However, whether or not it is required at every gene remains unclear.

Trans-acting factor dynamics at the poly(A) site affect transcription termination

Transcription termination of protein-coding genes requires recognition of the polyadenylation signal. This provides a signal for binding many factors involved in 3′-end formation, which in turn renders RNAPII competent for termination (Whitelaw and Proudfoot 1986; Logan et al. 1987; Connelly and Manley 1988; Birse et al. 1997; West et al. 2008). In yeast, mutations and conditional depletion of factors involved in pre-mRNA cleavage and polyadenylation, including homologs of CstF-64 (Rna15), CstF-77 (Rna14), Pcf11, CPSF160 (Yhh1), CPSF-73 (Ysh1), and Ssu72, result in read-through at the 3′-end of protein-coding genes (Birse et al. 1998; Dichtl et al. 2002; Steinmetz and Brow 2003; Garas et al. 2008). Accordingly, ChIP assays suggest that 3′-end processing factors, including Pcf11, Rna14, and Rna15, become associated with the RNAPII EC at the poly(A) site, in a Ser2 phosphorylated CTD-dependant manner (Ahn et al. 2004; Kim et al. 2004a; Luo et al. 2006). Other components of the yeast polyadenylation machinery, including homologs of CPSF-100, CPSF-30, and symplekin, are also associated with the 3′-ends of protein-coding genes (Kim et al. 2004a). ChIP analysis in human cells also revealed the association of components of the 3′-end processing complex, including CPSF-73 and CstF-64 at the 3′-end of genes (Swinburne et al. 2006; Glover-Cutter et al. 2008). Interestingly, mutations in RNAPII subunits Rbp3 and Rpb11 induce read-through at an mRNA and snoRNA terminator, leading Steinmetz et al. (2006a) to propose that a “termination signal” is transduced through the Rpb3/Rpb11 heterodimer. An intriguing possibility is that the “termination signal” involves interactions with 3′-end processing factors (Fig. 1).

While some 3′-end processing factors and RNAPII itself behave as termination factors, other proteins function as anti-terminators. In ChIP experiments, factors such as the Paf1C and the TREX/THO complex, which functions in mRNP biogenesis and maintenance of genome integrity (Garcia-Rubio et al. 2008), cross-link throughout genes, but their levels are reduced on passage through the poly(A) site, suggesting they are released from the EC (Ahn et al. 2004; Kim et al. 2004a). Dissociation of a CstF64-interacting protein, the transcriptional coactivator PC4 (human homolog of Sub1), at the poly(A) site renders RNAPII competent for termination, and genetic evidence in yeast suggests that Sub1/PC4 has an evolutionarily conserved anti-terminator activity (Calvo and Manley 2001). The yeast mRNA-binding protein Npl3 is thought to compete for mRNA binding with cleavage and polyadenylation factors, thereby preventing termination until transcription of the poly(A) site (Bucheli and Buratowski 2005; Bucheli et al. 2007). It is intriguing to note that Npl3, like p54nrb/PSF, plays multiple functions in pre-mRNA metabolism (Dermody et al. 2008; Kress et al. 2008), and all three contain related RNP-type RNA-binding domains.

Pcf11 activity may be particularly important for transcription termination. Pcf11 interacts with nascent RNA, contains a conserved CTD-interacting domain (CID), and mutations or depletion of Pcf11 cause defects in cleavage and termination (Licatalosi et al. 2002; Sadowski et al. 2003; Meinhart and Cramer 2004; Zhang et al. 2005). Using purified yeast RNAPII ECs, Zhang et al. (2005) showed that the CID of Pcf11 bridges the CTD to pre-mRNA, and can dismantle the EC in vitro, although this activity is independent of its activity in cleavage, and its physiological significance is unclear (Sadowski et al. 2003; Zhang et al. 2005). In Drosophila, depletion of Pcf11 causes transcriptional read-through, and Drosophila Pcf11 can also dismantle the EC in vitro (Zhang and Gilmour 2006). Zhang et al. (2007) also provided evidence that Pcf11 causes premature termination of an HIV provirus by limiting RNAPII processivity. However, in yeast, Pcf11 mutants defective in cleavage also showed transcriptional read-through of the ADH1 and PMA1 genes (Kim et al. 2006). In contrast to the previous experiments, this result argues that Pcf11 function in cleavage is necessary for efficient termination. Furthermore, depletion of Pcf11 in human cells by siRNA reduced the efficiency of RNAPII termination and stabilized the 3′-product of pre-mRNA cleavage, suggesting a role of Pcf11 in degradation of the 3′ RNA (West and Proudfoot 2008). These experiments together suggest that Pcf11 may also facilitate Xrn2-mediated degradation of the downstream RNA and efficient transcription termination, but the mechanism remains unclear.

Many factors involved in 3′-end formation and termination also associate with promoter regions (Calvo and Manley 2003). This has been observed in yeast (Bentley 2002; Calvo and Manley 2003; He et al. 2003) and humans (Dantonel et al. 1997; Venkataraman et al. 2005; Swinburne et al. 2006; Zhang et al. 2007; Glover-Cutter et al. 2008; Rozenblatt-Rosen et al. 2009), and suggests a cooperation between the transcription initiation and 3′-end processing/termination machineries. One scenario is that this facilitates recruitment of polyadenylation factors to active genes, and that these factors travel with the EC during transcription (e.g., Dantonel et al. 1997). Alternatively, studies in yeast suggest that gene looping brings promoter and terminator regions together during early stages of transcription, facilitating RNAPII reinitiation (O'Sullivan et al. 2004; Ansari and Hampsey 2005; Singh and Hampsey 2007). Moreover, these studies showed that gene looping is dependent on components of the cleavage/polyadenylation machinery and the general transcription factor TFIIB. In mammals, gene looping has been proposed to regulate transcription of the human BRCA1 gene (Tan-Wong et al. 2008). Additionally, Perkins et al. (2008) showed the existence of gene looping at an integrated HIV-1 provirus, which required a functional poly(A) signal.

Cooperation between the splicing machinery and termination might also take place. An intriguing study in C. elegans showed that an SR protein, SRp20, is implicated in mRNA transcription termination. Cui et al. (2008) propose that SRp20 functions either in degradation of the cleaved 3′-product or release of RNAPII from the template. ChIP assays combined with high-density microarrays showed that the splicing factor polypyrimidine tract-binding protein (PTB) is found at the 3′-ends of many genes and in some cases many kilobases past the mature 3′-end of the mRNA (Swinburne et al. 2006). However, PTB, a splicing repressor, is a multifunctional protein that also functions, for example, in 3′-end formation (e.g., Moreira et al. 1998). Thus, further experiments will be needed to confirm the role of PTB, and SRp20, in termination, and whether this, in fact, reflects a link with splicing. Notably, West and Proudfoot (2009) showed that termination enhances gene expression by promoting more efficient pre-mRNA processing, including post-transcriptional splicing.

Yeast sno/snRNAs

The majority of snoRNAs fall into two structurally and functionally well-defined classes. SnoRNAs carrying the conserved box C and D elements function mainly as guides in the site-specific 2′-O-methylation of target RNAs, whereas box H/ACA snoRNAs direct pseudouridylation. While some snoRNAs are required for pre-rRNA processing, the majority of snoRNAs guide modifications of pre-rRNAs (Kiss 2002). In mammals, these latter snoRNAs are encoded within introns of pre-mRNAs (Richard and Kiss 2006), while snoRNAs involved in pre-rRNA processing in humans, and most of the guide RNAs expressed in yeast, are transcribed by RNAPII from their own promoters.

Following cleavage by the endoribonuclease Rnt1 (yeast RNase III) (Chanfreau et al. 1998) or release from introns, the 3′-ends of snoRNA precursors are matured by exonucleolytic trimming via the exosome. In some cases, independently transcribed snoRNAs do not possess the RNA hairpin structures that are recognized by Rnt1 and may undergo endonucleolytic cleavage by the mRNA 3′-end formation machinery (Fatica et al. 2000; Morlando et al. 2002). After cleavage, cotranscriptional association of specific factors and formation of sn/snoRNPs protect the mature 3′-end of sn/snoRNAs from further exonucleolytic trimming (Morlando et al. 2004; Ballarino et al. 2005; Yang et al. 2005; Houalla et al. 2006).

Involvement of mRNA 3′-end processing factors in yeast snoRNA processing and termination

SnoRNAs and snRNAs are not polyadenylated. However, they require common factors from the cleavage/polyadenylation machinery (Fatica et al. 2000; Morlando et al. 2002), and thus cleavage must be uncoupled from polyadenylation. Cleavage factors are, in fact, required for correct termination and processing of sno/snRNAs (see Fig. 2; Morlando et al. 2002; Carneiro et al. 2008). Additionally, mutations in a subcomplex of the cleavage/polyadenylation machinery, the APT complex (associated with Pta1 [homolog of symplekin]), lead to read-through transcription of certain sn/snoRNA genes (Dheur et al. 2003; Ganem et al. 2003; Nedea et al. 2003; Steinmetz and Brow 2003; Kim et al. 2006; Ghazy et al. 2009). The APT factors implicated in sn/snoRNA termination include Pti1, a second yeast homolog of CstF-64 that also binds Rna14; Ref2, which interacts with the snoRNP-specific protein Nop1 (Morlando et al. 2004); the CTD phosphatase Ssu72 (Ganem et al. 2003; Steinmetz and Brow 2003; Kim et al. 2006); and Swd2, which is also a component of the Set1 complex (Cheng et al. 2004; Dichtl et al. 2004). Overexpression of Pti1 inhibits polyadenylation without affecting cleavage, suggesting that Pti1 may function in uncoupling cleavage and polyadenylation during snoRNA 3′-end formation (Dheur et al. 2003).

Open in a separate window

Figure 2.

Yeast RNAPII termination at snoRNA genes. Termination at terminator I (TI), the major termination site, is Nrd1-binding-dependent. The Nrd1 complex interacts with the Ser5-phosphorylated CTD and with the TRAMP and exosome complexes. The cleavage/polyadenylation machinery (CPF and CFIA) is not required for termination at TI (drawn as blurry complexes). Termination at terminator II (TII) involves cleavage/polyadenylation factors, while the Nrd1–TRAMP–exosome complex appears inactive in that process (blurry complexes). SnoRNA transcription couples termination with RNA surveillance. After release of the precursor, Trf4 and Pap1 adenylates the pre-snoRNA that will be processed by the exosome. Other factors shown to have a role in snoRNA termination are indicated. These include Pcf11, Paf1C, and the RNAPII subunits Rpb3 and Rpb11.

But the role of factors involved in 3′ processing/termination of pre-mRNA transcripts in snoRNA termination is not clear-cut. While ChIP experiments have shown that Rat1, mRNA cleavage factors including Rna15 and Rna14, as well as subunits of the APT complex are present at several snoRNA genes, surprisingly no termination defect was detected in RAT1 or RNA14 mutant strains (Kim et al. 2006). Normal snoRNA termination also occurred in strains lacking Rnt1 or the nuclear exosome component Rrp6 (Kim et al. 2006). Kim et al. (2006) also showed that cleavage by the cleavage/polyadenylation machinery is not required for snoRNA termination, as depletion of the CPSF-73 homolog Ysh1 or a mutation of PCF11 blocking cleavage did not affect snoRNA termination. However, transcription read-through was seen in strains expressing PCF11 mutants that are not capable of binding the CTD, and Garas et al. (2008) showed that termination at specific snoRNA genes is defective in a particular cold-sensitive YSH1 mutant strain. Interestingly, as shown in mRNA termination, Steinmetz et al. (2006a) observed that alteration of the heterodimer Rpb3/11 impairs snoRNAs termination. Another common point with mRNA termination is that in addition to proteins involved in 3′-end processing, the Paf1C is implicated in snoRNA termination. Defects in Paf1C lead to read-through transcription at snoRNA terminators (Sheldon et al. 2005). ChIP experiments suggested that Paf1C may facilitate snoRNAs 3′-end formation by recruiting Nrd1 (see below) (see Fig. 2; Sheldon et al. 2005).

The above results indicate that snoRNA genes appear to use different pathways to terminate transcription, as mutations or depletion in factors involved in mRNA termination have effects only on a subset of snoRNA genes. Further investigations will be needed to understand what underlies the differences among these genes.

The Nrd1 complex

Despite the apparent involvement of factors shared with pre-mRNA 3′-end processing, sno/snRNA termination relies heavily on a specific factor, the Nrd1 complex. Nrd1 (nuclear pre-mRNA down-regulation) is an essential nuclear RNA-binding protein that directs termination of snoRNA and some mRNA transcripts, in association with an interacting protein, the helicase Sen1 (Steinmetz and Brow 1996; Steinmetz et al. 2001; Ursic et al. 2004; Vasiljeva and Buratowski 2006). Nrd1 contains a CID (Meinhart and Cramer 2004; Meinhart et al. 2005) that interacts with CTD phosphorylated at Ser5 (Conrad et al. 2000; Gudipati et al. 2008; Vasiljeva et al. 2008a). Nrd1 also interacts with another essential RNA-binding protein, Nab3 (Yuryev et al. 1996).

Nrd1, Nab3, and Sen1 form the Nrd1 complex, which is detected by ChIP at snoRNAs genes (Kim et al. 2006) as well as some mRNAs genes, mainly at promoter regions (Nedea et al. 2003). Importantly, NRD1 and SEN1 mutations cause strong termination defects at some snoRNAs (Kim et al. 2006). Nrd1 and Nab3 recognize specific RNA sequences located downstream from the mature 3′-end of sn/snoRNAs (GUAA/G and UCUU, respectively) (Steinmetz and Brow 1996, 1998; Conrad et al. 2000; Steinmetz et al. 2001; Carroll et al. 2004, 2007), and mutations in these sites cause termination defects (Carroll et al. 2004). Several snoRNA terminators contain multiple potential binding sites for Ndr1 and Nab3, which form a heterodimer (Carroll et al. 2007). Nrd1 can also recruit the exosome complex, coupling the Nrd1 termination pathway to exosome-dependent trimming or degradation of transcripts (Fig. 2; Vasiljeva and Buratowski 2006).

Genome-wide analysis of RNAPII distribution in a SEN1 mutant strain confirmed the involvement of Sen1 in transcription termination of snoRNAs (Ursic et al. 1997; Rasmussen and Culbertson 1998) and other short transcripts (Steinmetz et al. 2006b). ChIP analysis showed RNAPII read-through at most snoRNAs genes in a SEN1 mutant strain or a strain defective for its helicase activity (Kim et al. 2006; Steinmetz et al. 2006b). Sen1 associates with the CTD and also coprecipitates with several snoRNAs and some snRNAs (Yuryev et al. 1996; Ursic et al. 1997, 2004). Nedea et al. (2008) showed that Glc7, the yeast PP1 (protein phophatase-1) catalytic subunit, is also a regulator of snoRNA termination, interacting directly with Sen1. Purification of TAP-tagged Sen1 identified Glc7, Nab3, Nrd1, and all the snoRNP core proteins as Sen1-associated proteins (Nedea et al. 2008). They propose that Sen1–Glc7–Nab3–Nrd1 form a stable complex in which Sen1 interacts directly with Nab3 and Glc7, which was shown to dephosphorylate Sen1 in vitro. Glc7 (He and Moore 2005) and PP1 in mammals (Shi et al. 2009) are known to function in mRNA 3′-end formation, and it will be of interest to elucidate the precise roles of dephosphorylation in all these processes.

Some snoRNA terminators appear to be bipartite, with a major (terminator I) Nrd1-binding-dependent terminator and a second, downstream terminator (terminator II) containing features similar to cleavage and polyadenylation signals (Fig. 2; Fatica et al. 2000; Morlando et al. 2002; Steinmetz and Brow 2003; Steinmetz et al. 2006a). Both terminators are required for efficient termination. Intriguingly, a snoRNA terminator can lead to polyadenylation when placed in the 3′ untranslated region (UTR) of a protein-coding gene, upstream of the natural poly(A) site (Steinmetz et al. 2006a). These data suggest that, depending on the gene context, snoRNA terminators possess the potential to direct polyadenylation. Previous data, indeed, reported polyadenylation of some sn/snoRNAs precursors in an exosome-defective yeast strain (van Hoof et al. 2000; Egecioglu et al. 2006). Accordingly, a strain deleted for RRP6 showed that one to four adenines are present at the 3′-end of C/D-box snoRNA precursors that had been incompletely processed (Grzechnik and Kufel 2008). Trf4/5 (PAPs from the TRAMP complex) add adenines at terminator I, while the canonical poly(A) polymerase, Pap1, polyadenylates snoRNA precursors at both terminators. SnoRNA read-through and defects in Nrd1 recruitment at terminator I in a TRAMP-defective strain indicate that the TRAMP complex is important for the appropriate association of Nrd1 with snoRNA genes and therefore for termination. Grzechnik and Kufel (2008) concluded that polyadenylation by Trf4 and Pap1 is a normal mechanism linked to snoRNA termination that stimulates 3′-end formation by the exosome (Fig. 2).

The regulation of transcription termination of snRNAs and snoRNAs involves a large number of factors, implicated in every step of transcription. Many factors are shared between mRNA and snoRNA termination pathways, and a tight regulatory network is necessary to correctly and efficiently discriminate between the two. An important feature of termination appears to be the distance traveled by RNAPII when it reaches the terminator. The Nrd1 complex is implicated in transcription of short transcripts (Steinmetz et al. 2006b). As explained below, this regulation reflects the different stages of CTD phosphorylation. On the other hand, the potential polyadenylation of snoRNAs adds another degree of complexity in possibly linking RNA surveillance, processing, and termination, and will need to be more fully explored.

Coupling of rRNA processing and termination

The 3′-end of the primary rRNA transcript is cleaved by the dsRNA-specific RNase III, Rnt1 (Elela et al. 1996; Kufel et al. 1999; Henras et al. 2004). Inactivation of Rnt1 causes read-through at T1, indicating that, as with protein-coding genes, cotranscriptional cleavage is important for efficient termination (Reeder et al. 1999; Prescott et al. 2004; Kawauchi et al. 2008). Recently, Catala et al. (2008) showed that Rnt1 interacts with two RNAPI-specific subunits, Rpa12 and Rpa34. Rpa12 shares sequence similarity with the small RNAPII subunit Rpb9 and with C11 of RNAPIII. While C11 is implicated in RNAPIII reinitiation (see below), Rpb9 might regulate arrest of elongating RNAPII at pause sites (Awrey et al. 1997). TRO analysis and electron microscopy (EM) visualization of Miller spreads demonstrated the involvement of Rpa12 in RNAPI termination (Prescott et al. 2004; Kawauchi et al. 2008). These experiments link transcription termination and processing of pre-rRNA. Deletion of Rnt1 or mutation disrupting its interaction with RNAPI altered the chromatin conformation of rDNA by increasing the number of open rRNA gene repeats. However, deletion of RPA12 alone had no effect on chromatin opening and pre-rRNA processing (Catala et al. 2008). Interestingly, as shown for RNAPII termination (Alen et al. 2002), chromatin remodeling factors are also involved in RNAPI termination. Deletion of CHD1, ISW1, and ISW2 had no effect on transcription initiation or on accumulation of rRNA, but caused an RNAPI termination defect (Jones et al. 2007). It is thus possible that normal RNAPI termination requires a specialized chromatin structure directed by these remodeling factors.

RNAPIII: a terminator on its own?

Three classes of RNAPIII-transcribed genes exist (tRNAs/VA RNAs, 5S rRNA, and U6 snRNA), which each uses a characteristic promoter. Despite the use of different promoters, RNAPIII terminates transcription at a simple common consensus sequence at the 3′-end of genes. More than 25 years ago, Bogenhagen and Brown (1981) identified a nucleotide sequence composed of a cluster of T residues in the coding DNA strand, required for transcription termination of the Xenopus laevis oocyte 5S RNA gene. Later, Cozzarelli et al. (1983) showed that a purified RNAPIII unable to carry out transcription initiation of a 5S DNA was able to terminate accurately and efficiently by recognizing the simple T-rich consensus. This experiment provided early evidence that RNAPIII itself possesses transcription termination activity without requirement of associated factors.

In vitro transcription using RNAPIII lacking the RNAPIII-specific subunits C11, C37, and C53 (RNAPIIIΔ) can support transcription initiation but fails to terminate properly (Landrieux et al. 2006). Adding back the heterodimer C37/C53 was sufficient to allow recognition of the terminator and to correct the termination defect, but not for transcription reinitiation. It was proposed that the C37/C53 complex reduces the elongation rate of RNAPIIIΔ, allowing for an increased pausing time at the terminator elements, which leads to release of the transcript and RNAPIII (Fig. 5). C11 is an essential subunit that mediates the intrinsic RNA cleavage activity of RNAPIII (Whitehall et al. 1994) and shows homology with the RNAPII subunit Rpb9 and to RNAPII elongation factor TFIIS (Chedin et al. 1998). Landrieux et al. (2006) showed that C11 RNA cleavage activity is not required for correct recognition of the RNAPIII terminator elements and release of the newly synthesized transcript. However, C11 itself, but not its cleavage activity, is required for reinitiation (Chedin et al. 1998; Landrieux et al. 2006). These experiments strongly suggest that termination and cleavage of the RNA precursor are not coupled in the RNAPIII transcription process. Consistent with its role in termination, structural analysis of RNAPIII showed that the C37/C53 heterodimer is positioned at the outer end of the DNA-binding cleft, toward the front of the EC, allowing the complex to sense the incoming DNA (Fernandez-Tornero et al. 2007).

Open in a separate window

Figure 5.

RNAPIII termination. Most of the termination activity is triggered by RNAPIII itself. The subunits C37 and C53 are essential for termination. The heterodimer C37–C53 might play a role in reducing the elongation rate of RNAPIII, allowing for an increased pausing time at the terminator, composed of a stretch of four to five Ts in the coding DNA strand. Several auxiliary factors, such as La, PC4, Topoisomerase I, or TFIIIC, are proposed to participate in RNAPIII termination in mammals.

Most RNAPIII termination signals occur within the first 40 bp downstream from the mature 3′-end of RNAs. In yeast, the most frequent T stretch length is six or seven with no stretch shorter than five Ts (Allison and Hall 1985; Hamada et al. 2000). In mammals, a stretch of four Ts is the most frequent terminator, and a stretch of more than five Ts is rare (Braglia et al. 2005). RNAPIII pauses not only at the terminator region but also at several other T stretches during transcription (Campbell and Setzer 1992; Matsuzaki et al. 1994). Campbell and Setzer (1992) proposed that RNAPIII release at the terminator is dependent on nascent RNA strand displacement during transcription elongation and can be uncoupled from termination signal recognition. RNAPIII recycling is also dependent on a natural termination signal (Dieci and Sentenac 1996; Ferrari et al. 2004). Termination can be influenced by sequences surrounding the T tract (Bogenhagen and Brown 1981; Mazabraud et al. 1987; Gunnery et al. 1999; Braglia et al. 2005). Braglia et al. (2005) proposed that the surrounding residues and, in particular, downstream residues of the yeast terminator influence the pausing time or the processivity of RNAPIII. However, no consistent pattern for this context effect has been elucidated.

We thank Emanuel Rosonina for his helpful corrections and comments on the manuscript and his support. P.R. is supported by a grant from the Muscular Dystrophy Association and relevant work in our laboratory is supported by grants from the NIH.