The Cid1 family of non-canonical poly(A) polymerases

YeastYeast 2006; 23: 991–1000.Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/yea.1408

Review

The Cid1 family of non-canonical poly(A) polymerasesAbigail L. Stevenson and Chris J. Norbury*Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

*Correspondence to:Chris J. Norbury, Sir WilliamDunn School of Pathology,University of Oxford, South ParksRoad, Oxford OX1 3RE, UK.E-mail:[email protected]

Received: 14 June 2006Accepted: 19 July 2006

AbstractPolyadenylation is an essential processing step for most eukaryotic mRNAs. In thenucleus, poly(A) polymerase adds poly(A) tails to mRNA 3′ ends, contributing totheir export, stability and translatability. Recently, a novel class of non-canonicalpoly(A) polymerases was discovered in yeast, worms and vertebrates. Differentmembers of the Cid1 family, named after its founding member in the fission yeastSchizosaccharomyces pombe, are localized in the nucleus and the cytoplasm and arethought to target specific RNAs for polyadenylation. Polyadenylation of a targetRNA by a Cid1-like poly(A) polymerase can lead to its degradation or stabilization,depending on the enzyme involved. Cid1-like proteins have important roles in diversebiological processes, including RNA surveillance pathways, DNA integrity checkpointresponses and RNAi-dependent heterochromatin formation. Copyright 2006 JohnWiley & Sons, Ltd.

Keywords: poly(A) polymerase; Schizosaccharomyces pombe; RNA stability; trans-lational control; Cid1 family; Trf4

Contents

IntroductionNuclear non-canonical PAPsCytoplasmic non-canonical PAPsThe CID1 family is conserved in higher eukaryotesConcluding remarksReferences

Introduction

The canonical poly(A) polymerase (PAP) is anessential nuclear enzyme, required for the generalpolyadenylation of mRNAs. In the past few years, anew family of PAPs has emerged that regulate spe-cific RNAs in both the nucleus and the cytoplasm.The family is named after Cid1, the first memberof this class of non-canonical PAPs discovered infission yeast [50]. Cid1 is a cytoplasmic enzymeinvolved in the response to replication stress that,unexpectedly, was found to possess poly(A) poly-merase activity [38]. Cytoplasmic polyadenylationis a well-known mechanism of gene regulation in

metazoan oocytes and neurons, but had not beendescribed previously in yeast.

Apart from Cid1, five other Cid1-like proteinswere discovered in Sz. pombe. Further sequenceanalysis revealed that Cid1 families are conservedthroughout eukaryotes [38,50]. All members ofthe Cid1 family are related to the canonical PAP,containing similar catalytic NTP transferase andPAP-associated domains. However, outside theseregions, Cid1-like proteins are highly divergedfrom the classical PAP, forming a novel class ofenzymes (Figure 1). Recent publications suggestthat all members of the Cid1 family are likely tobe PAPs; however, not all are cytoplasmic (sum-marized in Table 1). This review will explore thediverse biological roles played by both nuclearand cytoplasmic Cid1-like proteins in fission yeastand indicate how regulation of gene expressionby these enzymes is likely to be of great impor-tance in higher eukaryotes. After a discussion ofgeneral polyadenylation by the canonical PAP, thenuclear Cid1-like PAPs, Cid14 and Cid12, will bedescribed, followed by an insight into the cytoplas-mic enzymes, Cid1 and Cid13.

Copyright 2006 John Wiley & Sons, Ltd.

992 A. L. Stevenson and C. J. Norbury

Figure 1. Cid1-like proteins are conserved across eukaryotes. The Cid1 family are all members of the polymerase β familyof NTP transferases, which includes poly(A) polymerase (pink). All Cid1-like proteins share the catalytic NTP transferase(turquoise) and PAP-associated domains (red) but are otherwise highly diverged from the classical poly(A) polymeraseenzyme. The predicted size in amino acids of each protein is annotated. Hs7 is thought to be alternatively spliced to formHs7a/b (C.J.N., unpublished data). RRM, RNA recognition motif

Table 1. Summary of poly(A) polymerase enzymes (PAPs) in Sz. pombe

PAP Localization Known orthologues Function

Pla1 Nuclear Pap1p (S.c.), PAP (H.s.) Essential mRNA 3′ processing enzymeCid1 Cytoplasmic CID-1 (C.e.) Checkpoint response to replication blockCid11 Nuclear and cytoplasmic ? ?Cid12 Nuclear and cytoplasmic RDE-3 (C.e.) Required for RNAi-dependent transcriptional silencingCid13 Nuclear and cytoplasmic ? Maintains dNTP pools via ribonucleotide reductaseCid14 Nuclear/ nucleolar Trf4/5p (S.c.) Targets RNAs for exosome-mediated degradationCid16 Mitochondrial? hmtPAP (H.s.)? Mitochondrial RNA processing?

S.c., Saccharomyces cerevisiae; H.s., Homo sapiens; C.e., Caenorhabditis elegans.

Polyadenylation by the canonical poly(A)polymerase, PAP

Polyadenylation is an essential processing step foralmost all eukaryotic mRNAs, with the notableexception of metazoan bulk histone mRNAs. The 3′end of a pre-mRNA is polyadenylated in a two-stepreaction, where endonucleolytic cleavage is fol-lowed by poly(A) addition. Like many RNA pro-cessing reactions, polyadenylation occurs while the

mRNA is being transcribed by RNA polymeraseII (pol II). Excellent reviews exist on the sub-ject [35,58], so only a brief description is outlinedbelow.

Polyadenylation is a complex reaction requiringmany multi-subunit protein complexes and severalcis-acting sequences, which were first described inmammalian cells, but functional equivalents alsoexist in yeast. In mammals, two sequence elementsflank the cleavage site and direct poly(A) addition.

Copyright 2006 John Wiley & Sons, Ltd. Yeast 2006; 23: 991–1000.DOI: 10.1002/yea

Non-canonical PAPs 993

The hexanucleotide signal sequence AAUAAA(or sometimes a variant of this sequence) isfound 10–30 nucleotides (nts) upstream of thecleavage/poly(A) site and is highly conserved inanimals [36]. A more variable GU-rich downstreamelement (DSE) is located 20–40 nts below thecleavage site [26]. The sequence variability anddistance between these two elements determinethe strength of the poly(A) signal and specify thesite of cleavage, which generally occurs after aCA dinucleotide [6]. The hexanucleotide sequenceis recognized by the 160 kDa subunit of CPSF(cleavage and polyadenylation specificity factor)and the DSE is bound by the 64 kDa subunit ofCstF (cleavage stimulation factor) [22,30]. CPSFtakes part in both the cleavage and polyadeny-lation reactions, helping to recruit PAP [30]. Onthe other hand, CstF is required only for thecleavage reaction and together with CPSF, recruitsthe additional cleavage factors, CFIm and CFIIm[44]. Assembly of the polyadenylation machin-ery also requires the carboxy-terminal domain ofRNA pol II and the scaffold protein, symplekin[14,43].

By comparison with the situation in mam-mals, the yeast polyadenylation machinery is lesswell defined. In the budding yeast, Saccharomycescerevisiae, two variable upstream elements areidentifiable, an AU-rich efficiency element (EE)and an A-rich positioning element (PE). Flank-ing the poly(A) site are two U-rich sequences,the upstream sequence element (USE) and thedownstream sequence element (DSE) [7]. Mam-malian and yeast polyadenylation complexes con-tain functionally orthologous components, but alsopossess unique factors. Three complexes exist inbudding yeast: cleavage polyadenylation factor(CPF), cleavage factor IA (CFIA) and cleavagefactor IB (CFIB) [35]. CPF contains many fac-tors functionally homologous to CPSF and sym-plekin, but also includes the poly(A) polymerase,Pap1p. The subunit composition of CFIA is sim-ilar to that of CstF and CFIIm. The yeast CFIBcomplex, an RNA-binding protein called Hrp1p,has no known homologue in mammalian cells[19]. The mechanism of polyadenylation in Sz.pombe has not been studied in great detail, butone study suggests that mRNA 3′ end forma-tion in fission yeast is broadly similar to thatin budding yeast [16]. The canonical PAP in Sz.

pombe is encoded by pla1, which can function-ally replace its S. cerevisiae equivalent, PAP1[33].

The newly-formed poly(A) tail is immediatelybound by poly(A) binding protein (PABP; reviewedin [23]). Nuclear PABPs regulate the ultimatelength of the poly(A) tail and are required for somemRNAs to be exported from the nucleus. In thecytoplasm, PABPs facilitate the formation of the‘closed loop’ mRNP structure that is crucial forpromoting translation initiation and termination, therecycling of ribosomes and increasing mRNA sta-bility [52].

Cytoplasmic polyadenylation

In addition to the essential 3′ end processing reac-tion that occurs in the nucleus, mRNAs can bepolyadenylated in the cytoplasm. The mechanismof cytoplasmic polyadenylation is distinct from thatin the nucleus and has been well characterized inanimal oocytes and neuronal cells (reviewed in[26]). This process requires some of the factorsessential for nuclear polyadenylation, but also spe-cific factors required for cytoplasmic polyadenyla-tion, such as cytoplasmic polyadenylation elementbinding protein (CPEB). In general, extension ofpoly(A) tails in the cytoplasm leads to translationalactivation, whereas deadenylation causes transla-tional repression [42]. This mechanism of trans-lational control allows for a rapid upregulation ingene expression, without the need for new tran-scription.

Gene expression in vertebrate oocytes is con-trolled principally at the level of translation, wherematernal mRNAs are kept dormant until later indevelopment, when they are translationally acti-vated by cytoplasmic polyadenylation. Cytoplasmicpolyadenylation was initially thought to regulategene expression only during early development;however, there is increasing evidence that it alsohas a role to play in somatic cells, such as neu-rons. In the mouse, CPEB-mediated polyadeny-lation is required for the activation of dendriticmRNAs in neurons, in response to synaptic stimu-lation [56].

The PAP responsible for cytoplasmic polyadeny-lation in animal oocytes was not identified untilrecently. Evidence has emerged to suggest that aCid1-like protein is the cytoplasmic PAP recruited



by CPEB [1]. However, since CPEB is not con-served in fission yeast, the mechanism of Cid1-mediated cytoplasmic polyadenylation in Sz. pombeis likely to differ from that described in early ani-mal development. The biological functions both ofnuclear and of cytoplasmic Cid1 family proteinsare discussed below.

Nuclear non-canonical PAPs

Cid14 and the TRAMP complex

Cid14 is a nuclear enzyme, enriched in the nucle-olus [54]. There is strong evidence that Cid14 is akey component of the nuclear RNA surveillancepathway, aiding the multi-catalytic ribonuclease,the exosome, to degrade aberrant RNA species.Much of this evidence comes from studies of theS. cerevisiae homologues of Cid14 and Trf4/5p.

S. cerevisiae Trf4/5p

Only two Cid1-related proteins exist in buddingyeast, Trf4p and Trf5p. In contrast to Cid1, bothproteins are nuclear [47]. The lack of a cytoplas-mic Cid1-like protein and the reduced number ofparalogues suggest that budding yeast lack some ofthe cellular processes shared by fission yeast andhigher eukaryotes. TRF4 was originally identifiedas a gene that, when mutated, showed syntheticlethality with mutations in DNA topoisomerase I(TOP1 ), an enzyme that relaxes the supercoiling

of the DNA duplex, allowing replication to pro-ceed [40]. Further genetic studies identified a TRF4paralogue, TRF5 ; although neither gene is essen-tial on its own, a trf4∆trf5∆ double mutant isnon-viable, implying that Trf4p and Trf5p haveoverlapping, essential functions [4]. Further anal-yses indicated a mitotic role for Trf4p; Trf4p inter-acts with a subunit of cohesin, Smc1, and a trf4-tstop1∆ mutant is defective in nuclear segregationat the restrictive temperature [3]. Confusingly, evi-dence also emerged for an essential role for Trf4pin DNA replication; Trf4p purified from bacteriawas reported to have DNA polymerase activityin vitro, trf4-ts trf5∆ cannot complete S-phase atthe restrictive temperature and Trf4p can interactwith and stimulate DNA pol ε [10,51]. Together,these studies led to the notion that Trf4/5p is thefourth essential DNA polymerase in budding yeast,DNA pol σ , required for the establishment of sisterchromatid cohesion [2,51].

Recently, the role of Trf4/5p as a DNA poly-merase has come into question, with the publi-cation of numerous independent reports of Trf4ppossessing PAP activity [21,46,57]. These studiesalso elucidated a biological role for Trf4p-mediatedpolyadenylation in the activation of the nuclearexosome. The nuclear exosome is a complex ofmultiple 3′ –5′ exonucleases, required for the degra-dation and/or processing of numerous RNAs [27].However, the purified exosome has only weakribonuclease activity in vitro, suggesting that other

Figure 2. The nuclear non-canonical poly(A) polymerases Cid12 and Cid14. (A) Cid12 acts with Hrr1 and Rdp1 to formthe RDRC complex (red), which, along with the RITS complex (blue), directs RNAi-dependent heterochromatic genesilencing. (B) Cid14 is homologous to Trf4p, part of the TRAMP complex, which, along with Air1/2p and Hrr1p, targetsRNAs for degradation by the nuclear exosome (shown in pink)



co-factors are necessary for efficient RNA degra-dation in vivo. Trf4p was found to be a compo-nent of the TRAMP complex, which can stimu-late the activity of the exosome in vitro and isrequired for the processing of numerous exosomesubstrates in vivo [21,46]. Trf4p interacts with anRNA helicase (Mtr4p) and one of two functionally-redundant putative RNA-binding proteins (Air1p orAir2p) to form the Trf4/Air1/Mtr4 polyadenylation(TRAMP) complex. In this new model (Figure 2B),Air1/2p recruits the TRAMP complex to its tar-get RNAs, which are oligo-adenylated by Trf4p.The oligo(A) tract allows recruitment and activa-tion of the exosome, which degrades the RNAin a 3′ –5′ direction, facilitated by the helicaseactivity of Mtr4p. It is likely that multiple roundsof TRAMP oligo-adenylation and exosome degra-dation are required for complete destruction ofthe RNA.

The original report of Trf4p as an activa-tor of exosome-mediated degradation describedan aberrantly-modified initiator tRNA (tRNAMet)as a substrate for Trf4p oligo-adenylation [17].Since then, Trf4p has been shown to targetvarious RNAs for degradation and/or process-ing in the nucleus, including pre-rRNAs, pre-snoRNAs and intergenic cryptic transcripts [21,57].However, these studies failed to show Trf5ppoly(A) polymerase activity in vitro. Since then,several groups have been successful in purifyingactive Trf5p and have shown that it also con-tributes to rRNA processing, like Trf4p [11,13,15].Together, these results suggest that Trf4/5p and theTRAMP complex are part of a nuclear surveil-lance mechanism in eukaryotes, preventing theexport and subsequent expression of aberrantRNAs.

The conflicting reports describing Trf4p enzy-matic activity are difficult to reconcile, since itis unlikely that Trf4p possesses both DNA poly-merase and poly(A) polymerase activities. Giventhe solid evidence presented in the recent papersdescribing the TRAMP complex and the aminoacid sequence similarity between Trf4/5p and therest of the Cid1 family, it is more likely that Trf4/5pare poly(A) polymerases.

Sz. pombe Cid14

A recent publication established that Cid14 is theSz. pombe homologue of Trf4/5p [54]. Unlike

Trf4/5p, Cid14 is non-essential, but a cid14∆ strainhas a slow growth phenotype, aberrant nucleo-lar structure and suffers from chromosome seg-regation defects. The expression of cid14 + cancomplement the temperature sensitivity of a trf4-ts top1∆ mutant, whereas other Sz. pombe cidgenes cannot. In addition, cid14∆ has defects inpre-rRNA processing, like trf4∆, and polyadeny-lated rRNA accumulates in an exosome mutant ina Cid14-dependent manner. Although Cid14 PAPactivity has not yet been confirmed in vitro, theseresults indicate that, like Trf4/5p, Cid14 is likelyto polyadenylate pre-rRNAs to target them for pro-cessing by the exosome. This suggests that themechanism of exosome activation by polyadenyla-tion described in S. cerevisiae is conserved in othereukaryotes.

Cid12 and heterochromatin formationAn unexpected role for a Cid1-like protein in hete-rochromatin formation was discovered when Cid12was identified as part of the RNA-directed RNApolymerase complex (RDRC), which also includesthe RNA-directed RNA polymerase Rdp1 and theRNA helicase Hrr1 [29]. RNAi-dependent het-erochromatin formation in Sz. pombe (reviewedin [24]) requires the RDRC and RITS (RNA-induced transcriptional silencing) complexes. Inbrief, short interfering RNAs (siRNAs) producedby Dcr1 from centromeric transcripts are loadedinto the RITS complex (containing Ago1, Tas3and Chp1). Together, RDRC and RITS directcentromeric histone methylation by Clr4, lead-ing to Swi6 and cohesin binding and the for-mation of transcriptionally-silent heterochromatin(Figure 2A). cid12∆ mutants are defective incentromeric silencing and have severe chromo-some segregation defects in both mitosis andmeiosis, providing further evidence for an essen-tial role in heterochromatin formation [29,55].Also, in cid12∆ mutants, the RITS complex isdevoid of all siRNAs [29]. The polyadenylationactivity of Cid12 is likely to be important forthis function, since mutation of residues requiredfor PAP activity also results in loss of cen-tromeric silencing [55]. However, the role of theCid12-generated poly(A) tail and its RNA tar-gets are yet to be characterized. In keeping withits proposed role in RNAi, Cid12 was local-ized mainly to the nucleus by immunofluores-cence, but some cytoplasmic localization was also



observed [29]. Cid12 may have other roles, in addi-tion to and independent from its RNAi function.Cid12 interacts with numerous spliceosome pro-teins, as well as the RDRC [29] and genetic anal-ysis of cid12∆ revealed a disrupted S–M check-point phenotype when it was combined with DNApolymerase mutants, like that seen with cid1∆

[55].

Cytoplasmic non-canonical PAPs

Cid1 is a cytoplasmic poly(A) polymeraseinvolved in the replication checkpoint

An overexpression screen for novel componentsof the fission yeast replication checkpoint identi-fied a previously uncharacterized gene, cid1 + (forcaffeine induced death suppressor) [49]. A geneticstudy provided further evidence that Cid1 wasinvolved in the checkpoint response to replicationstress [50]. Deleting cid1 caused sensitivity to acombination of hydroxyurea (HU) and caffeine andcid1 + overexpression partially suppressed the HU-sensitivity of ‘checkpoint rad’ mutants, includingrad3∆, rad9∆ and rad17∆. Also, DNA pol δ andε mutants are checkpoint-defective in the absenceof Cid1; cdc27-P11 (pol δ mutant) and cdc20-P7 (pol ε mutant) have intact S–M checkpoints,which are partially disrupted in cid1∆ mutants.This study also determined that Cid1 is likely toact through Chk1, since cds1∆cid1∆ cells have anadditive phenotype, similar to that of cds1∆chk1∆

cells upon HU treatment. HU-treated cds1∆ andDNA polymerase mutants both depend on Chk1to prevent mitotic entry [12]. Therefore, Cid1 maycontribute to a Chk1-dependent sub-pathway of theS–M checkpoint in Sz. pombe.

A biochemical investigation determined thatCid1 was not a DNA polymerase, but a PAPin vitro; Read et al. [37] determined Cid1 to beexclusively cytoplasmic in vivo and to target asubset of RNAs in vitro. Microarray studies havealso shown that Cid1 does not target mRNAs inunperturbed cells, but alters the levels of a subsetof RNAs in response to replication stress in vivo(A. Stevenson, unpublished data). Therefore, it wasproposed that Cid1 is a novel type of cytoplasmicpoly(A) polymerase important for cell cycle regu-lation [38]. It was hypothesized that Cid1 upregu-lates the expression of specific genes important for

checkpoint control by targeting their mRNAs forpolyadenylation in the cytoplasm, increasing theirstability and/or translatability (Figure 3).

Cid13 affects RNAs in response to hydroxyurea

Two independent studies identified Cid13 asanother cytoplasmic PAP of Sz. pombe [37,41].However, a recent global protein localization studyin fission yeast revealed that Cid13 fused to yel-low fluorescent protein (YFP) localizes to boththe nucleus and the cytoplasm [25]. Cid13 wasoriginally identified as a multi-copy suppressorof the HU-sensitivity of rad3-ts and rad26-∆N73mutants. Cid13 purified from yeast has PAP activ-ity in vitro but, unlike Cid1, Cid13 is likely tofunction in a checkpoint-independent manner toallow cells to survive replication stress. A cid13∆

strain is hypersensitive to HU treatment as a resultof reduced cellular levels of dNTPs and over-expression of cid13 + cannot suppress the S–Mcheckpoint defect of rad3∆. Therefore, Cid13 wasproposed to act by restoring dNTP levels, butthe mechanism of how Cid13 achieves this bycytoplasmic polyadenylation remains controversial.One study reported that Cid13 targets the cellcycle-regulated isoform of suc22 mRNA, whichencodes the small subunit of ribonucleotide reduc-tase (RNR) [41]. In this model, Cid13-mediatedcytoplasmic polyadenylation of suc22 increases itsstability and translatability, which enhances dNTPsynthesis. In support of this, a subtle decrease in

Figure 3. Cid1 is a cytoplasmic non-canonical poly(A)polymerase. Cid1 is activated in response to replicationblock, targeting specific mRNAs (pink) for polyadenylationin the cytoplasm. This is likely to lead to increasedtranslatability, allowing rapid upregulation of key proteins,necessary for the appropriate response to replication stress



the abundance, stability and polyadenylation of oneform of suc22 mRNA was detected in cid13∆mutants. However, another group found no reduc-tion in Suc22 protein levels in cid13∆ cells, even inthe presence of HU [37]. Therefore, it is likely thatCid13 targets other RNAs in addition to suc22 tomaintain dNTP levels in response to HU treatment.

Other Sz. pombe Cid1-like proteins

Although Sz. pombe Cid11 and Cid16 are yetto be fully characterized, neither is required forcellular viability (S.W. Wang, unpublished results).Their strong sequence similarity to the confirmedPAPs, Cid1 and Cid13, suggest that they are alsolikely to possess a polyadenylation activity. In therecent global localization study by Yoshida and col-leagues, Cid11-YFP localized to both the nucleusand cytoplasm and Cid16-YFP localized to smallspeckles in the cytoplasm, which may be indicativeof mitochondrial staining [25]. In light of recentdata, it is unlikely that Cid1-like proteins performredundant functions in Sz. pombe. Therefore, it ispossible that each Cid1-like protein targets a dif-ferent set of RNAs to affect different cellular pro-cesses. A summary of the Cid1 family in Sz. pombeis described in Table 1. Encoding such a repertoireof regulatory poly(A) polymerases provides the fis-sion yeast cell with increased flexibility for thecontrol of gene expression, which is likely to be ofgreat importance in higher eukaryotes, as discussedbelow.

The CID1 family is conserved in highereukaryotes

Cid1 protein families are conserved from yeast tohumans (Figure 1). The first member of the Cid1family to be characterized in higher eukaryoteswas GLD-2, a cytoplasmic poly(A) polymerase ofthe nematode Caenorhabditis elegans [48]. gld-2 was identified as a gene involved in controlof germline development and promotes the tran-sition from mitosis to meiosis [18]. This studyalso identified a GLD-2 interacting partner, GLD-3,a bicaudal C-like KH domain RNA-binding pro-tein also required for germline development [9].Therefore, it was proposed that GLD-2 and GLD-3 form a heterodimeric PAP in vivo; GLD-3 maydirect GLD-2 to meiosis-specific mRNAs to allow

their cytoplasmic polyadenylation, stabilization andsubsequent translation [48]. Like fission yeast, C.elegans has numerous other Cid1-like proteins. Apotential Cid12 homologue, RDE-3, is essential forRNAi and fertility, supporting a central role forCid1-like proteins in gene silencing pathways [5].A recent publication describes CID-1, a memberof the Cid1 family in C. elegans important for thecheckpoint response to replication block [34]. Thisreport confirmed a role for Cid1-like proteins incheckpoint pathways and suggested a functionalhomologue for Cid1 in a multicellular organism.

In an attempt to identify novel factors respon-sible for cytoplasmic polyadenylation in Xenopuslaevis oocytes, Richter and colleagues discovereda GLD-2 homologue (xGLD-2) through its co-purification with symplekin [1]. In this elegantstudy, Barnard et al. presented strong evidence thatxGLD-2 is the long-sought enzyme responsible forcytoplasmic polyadenylation in metazoan oocytes.Together, these studies suggest that xGLD-2 is tar-geted to its mRNA substrates in the cytoplasm byCPSF and CPEB, with symplekin acting as a scaf-fold protein to promote these interactions, formingthe ‘G-complex’ (for GLD-2).

Five Cid1-like proteins were identified in humansand were named Hs1–Hs5 [20]; however, closerinspection of the annotated human genome revealsthe existence of at least seven human Cid pro-teins (Table 1). As a result of this study andtheir high sequence similarity to C. elegans GLD-2, Hs1 and Mm1 were renamed human GLD-2(hGLD-2) and murine GLD-2 (mGLD-2). Recom-binant murine and human GLD-2 were also shownto be active PAPs in vitro [32,39]. There is alsoindirect evidence that murine and human GLD-2 homologues are responsible for localized cyto-plasmic polyadenylation of neuronal mRNAs atsynapses; murine and human gld-2 mRNA is abun-dant in the brain and co-localizes with CPEB1and pumilio1 mRNAs in the hippocampus [39].Besides Hs1/hGLD-2, only one other Cid1-likeprotein has been characterized in human cells.Hs4 is a PAP localized to the mitochondria,also known as human mitochondrial PAP (hmt-PAP [31,45]; S. Min, unpublished results). In ani-mals, mitochondrial-encoded mRNAs (mtRNAs)are polyadenylated; this process is required to gen-erate complete stop codons for some mtRNAs [28].The enzyme responsible was not thought to be the



canonical PAP, which lacks a mitochondrial tar-get sequence. Recently, Hs4 was identified as themitochondrial PAP required for mtRNA polyadeny-lation; mtRNA targets of Hs4 include those encod-ing the oxidative chain enzymes, COXI-III, ATP6/8and ND3 [31,45]. Since Cid16 has been recentlylocalized to the mitochondria, perhaps Cid16 is afunctional homologue of hmtPAP in fission yeast[25]. As yet, a Trf4p/Cid14 homologue in highereukaryotes has not been described. However, itis likely that polyadenylation by a nuclear Cid1-like protein is also involved in exosome activationin humans, as a report of oligo-adenylated exo-some substrates in HeLa cells has been publishedrecently [53].

Concluding remarks

The Cid1 family describes an entirely novel groupof PAPs, with its members being evolutionarilydistinct from the canonical PAP, but having asimilar catalytic activity. Cid1-like proteins areregulatory PAPs, targeting specific RNAs to controlthe expression and/or processing of transcriptsinvolved in diverse cellular processes. Unlike thecanonical PAP, the majority of Cid1-like proteinslack an RRM; therefore, it is likely that additionalRNA-binding protein partners are required to targettheir substrates for polyadenylation, such as Sz.pombe homologues of Air1/2p, GLD-3 or CPSF.As well as bringing the PAP to the 3′ end of a targetRNA, such RNA-binding partners could providesequence specificity. In this way, Cid1-like proteinsmay interact with a number of binding partners,allowing them to target different substrates indifferent situations.

In general, polyadenylation by cytoplasmic mem-bers of the Cid1 family seems to be involvedin the stabilization and increased translatabilityof their target RNAs, whereas polyadenylation ofRNAs by nuclear Cid1-like proteins leads to theirdegradation by the nuclear exosome. If this is thecase, nuclear polyadenylation by Cid1-like proteinsis reminiscent of the situation in bacteria, wherepolyadenylation also targets RNAs for degradation[8]. Nuclear Cid1-like enzymes may have retainedthis bacterial-like ability to target RNAs for degra-dation, whereas PAP-dependent polyadenylationevolved to allow mRNAs to escape being targeted

by the nuclear exosome, perhaps by being boundby PABP.

Acknowledgements

Work in the authors’ laboratory is supported by the MedicalResearch Council, the Association for International CancerResearch and Cancer Research UK. A.L.S. is supported bythe MRC, Grant No. G78/7515.

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The Cid1 family of non-canonical poly(A) polymerases

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