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Planta (2014) 239:1159–1173DOI 10.1007/s00425-014-2058-6

RevIew

Functional conservation and divergence of J-domain-containing ZUO1/ZRF orthologs throughout evolution

Dong-Hong Chen · Yong Huang · Chunlin Liu · Ying Ruan · Wen-Hui Shen

Received: 18 November 2013 / Accepted: 5 March 2014 / Published online: 22 March 2014 © Springer-verlag Berlin Heidelberg 2014

terminal SANT domains, suggesting the divergence of their functions with those in fungi. Zuotin/Zuotin-related factor proteins retain the ancestral function as an Hsp70 co-chaperone implicated in protein folding and rena-turation after stress; these proteins also perform diverse neofunctions in the cytoplasm and transcriptional and/or epigenetic regulatory functions in the nucleus. There-fore, these proteins are involved in translational fidelity control, ribosomal biogenesis, asymmetric cell division, cell cycle, apoptosis, differentiation, and tumorigenesis. The results of sequence and domain organization analy-sis of proteins from diverse organisms provided valuable insights into the evolutionary conservation and diver-sity of ZUO1/ZRF protein family. Further, phylogenetic analysis provides a platform for future functional inves-tigation on the ZUO1/ZRF protein family, particularly in higher plants.

Keywords DnaJ · Hsp40 · J-domain · Phylogeny · SANT · ZUO1/ZRF

AbbreviationsCTD C-terminal domainGlsA Gonidialess AMIDA1 Mouse Id associated 1NBD N-terminal binding domainNeS Nuclear export signalPdr1 Pleiotropic drug resistance 1PRC1 Polycomb repression complex 1RAC Ribosome-associated complexSANT Swi3, Ada2, NcoR1 and TFIIIBSBD C-terminal binding domainUBD Ubiquitin-binding domainZRF ZUO1-related factorZUO1 Zuotin

Abstract Main conclusion Review and phylogenic analysis on ZUO1/ZRF orthologs.Abstract Heat shock protein 40s (Hsp40s), also known as J-proteins, are conserved in prokaryotes and eukary-otes. The Zuotin/Zuotin-related factor (ZUO1/ZRF) fam-ily belongs to a novel Hsp40 clade exclusively found in eukaryotes. Zuotin/Zuotin-related factor proteins are characterized by a large N terminal ZUO1 domain orig-inally identified in the yeast ZUO1 protein. The ZUO1 domain is characterized by a highly conserved J-domain, together with an atypical UBD domain first identified in the human ZRF1 protein. Furthermore, ZUO1/ZRF pro-tein families in animals and plants harbor a pair of C

D.-H. Chen and Y. Huang are contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2058-6) contains supplementary material, which is available to authorized users.

D.-H. Chen · Y. Huang · C. Liu · Y. Ruan Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Agricultural University, Changsha 410128, China

D.-H. Chen (*) · Y. Huang · Y. Ruan (*) Key Laboratory of education, Department of Hunan Province on Plant Genetics and Molecular Biology, Hunan Agricultural University, Changsha 410128, Chinae-mail: chendh212@163.com

Y. Ruan e-mail: yingruan@hotmail.com

w.-H. Shen Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France

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Introduction

Ribosomes in prokaryotes and eukaryotes perform a crucial function in the progress of decoding genetic information by translating mRNA to a primary amino acid sequence, which is subsequently folded to form an activated three-dimensional conformation. Specialized molecular chap-erones, such as different types of heat shock protein 40 (Hsp40)–Hsp70 complexes, are required to bind budding polypeptides and assist protein folding. Heat shock pro-tein 40 (J-proteins) predominantly function as co-chaper-ones of important molecular chaperone Hsp70s in diverse processes, such as protein translation, folding, unfolding, translocation, and degradation (Hennessy et al. 2005; Qiu et al. 2006). The activity and specificity of Hsp70 proteins can be regulated by different Hsp40 members, which stim-ulate the ATPase activity of Hsp70s and stabilize the bind-ing of client proteins to Hsp70s. Heat shock protein 40 pro-teins are highly conserved in prokaryotes and eukaryotes; several homologs, including 6 in Escherichia coli, 22 in Saccharomyces cerevisiae, 41 in human, 92 in Arabidopsis, and 83 in rice, have been identified (walsh et al. 2004; Qiu et al. 2006; Rajan and D’Silva 2009; Sarkar et al. 2013).

Heat shock protein 40 are characterized by the pres-ence of a highly conserved DnaJ domain (J-domain) which

is mainly responsible for the interaction with Hsp70. The J-domain is usually located at the N terminus and consists of four α-helices and a loop region harboring an extremely conserved tri-peptide (HPD motif) between helices II and III (Qian et al. 1996). Heat shock protein 40 are categorized into three classes based on the overall domain organiza-tion: types I, II, and III (Cheetham and Caplan 1998). Type I Hsp40 protein is found in E. coli with a single-member DnaJ and consists of three sequentially canonical domains: a J-domain; a Gly/Phe-rich region and a zinc-finger domain containing four repeats of the CxxCxGxG motif (x indicates any residue) at the N terminus; and an uncharacterized C terminal region (Fig. 1a). Type II protein, such as E. coli CbpA, only lacks a zinc-finger domain at the N terminus. Type III protein, including DjlA, DjlB, DjlC/Hsc56, and HscB/Hsc20 in E. coli (Fig. 1a), only contains a J-domain.

Based on the classification criterion of the Hsp40 fam-ily, S. cerevisiae Zuotin/ZUO1, in which zuo means left in Chinese (Zhang et al. 1992), belongs to type III proteins similar to DjlA, DjlB, DjlC, and HscB in E. coli. How-ever, DjlA shows a different distribution of J-domain at the C terminus (Fig. 1a). DjlB and DjlC contain glutamic acid (e) in place of aspartic acid (D) in conserved HPD residues (Fig. 1a); however, this observation is inconsistent with common J-domain characteristics (walsh et al. 2004).

Fig. 1 Phylogeny of J-proteins in E. coli and several typical ZUO1/ZRF proteins in eukary-otes. a Phylogeny of J-domains related to the three types of Hsp40 proteins in E. coli. Boot-strap support (500 replicates) based on the maximum likeli-hood method and JTT-substi-tuted model is indicated below the branches when it exceeds 50 %. Zf zinc-finger domain, Tm transmembrane region. b Detailed domain architecture of ZUO1 orthologs in several rep-resentative species. ZUO1/ZRF proteins are selected from Volvox vcGlsA, Arabidopsis atDjC1 (renamed as AtZRF1a), human ZRF1, mouse MIDA1, and yeast ZUO1. J J-domain, S1 SANT1, S2 SANT2, Z ZUO1 domain, ± charged domain, G G/F-rich region, Zf zinc-finger domain

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Phylogenetic analysis results based on the conserved J-domain have shown that ZUO1 is only grouped with its eukaryotic orthologs and not with the type III Hsp40s in E. coli (Fig. 1a). Type I Hsp40 HDJ2 in humans and YDJ1 in yeast are also grouped together and exhibit a relatively closer relationship with the group that includes type I DnaJ and type II CbpA in E. coli. Therefore, domain architec-ture can be used as an important reference for gene clas-sification but is insufficient to clarify the relationships among homologs. Moreover, ZUO1-like proteins should only exist in eukaryotes. A ZUO1 ortholog usually contains a structurally defined ZUO1 domain, which consists of a highly conserved J-domain and a ubiquitin-binding domain (UBD)/M domain, which was initially identified in the mammalian ZUO1 ortholog ZRF1/MIDA1 (Fig. 1b).

Zuotin-like proteins in eukaryotes except fungi conven-tionally contain a pair of closely juxtaposed SANT (Swi3, Ada2, NcoR1, and TFIIIB) DNA-binding domains at the C terminus. The SANT domain is often found in the components of some several chromatin remodeling com-plexes associated with the recruitment of histone acetylases or deacetylases (Aasland et al. 1996). SANT domain has highly structural similarity to the DNA-binding domain of Myb-related proteins (Grune et al. 2003). Both domains consist of three tandem α-helix repeats arranged in a helix-turn-helix motif, in which each α-helix contains a bulky aromatic residue (Fig. 4c) (Ogata et al. 1994; Tahirov et al. 2001). Despite the overall similarity, the SANT domain yields a relatively acidic pI and overall negatively charged surface or a non-acidic pI but contains large patches of acidic residues asymmetrically distributed on the sur-face; this property is for positively charged histone bind-ing but differs from the basic characteristic of a canonical Myb DNA-binding domain; the latter is inclined toward negatively charged DNA recognition and may cause func-tional divergence between the SANT domain and the Myb DNA-binding domain (Boyer et al. 2004). with these novel domains, ZUO1 orthologs can also perform neofunctions in different species.

DnaJ: potential ancestral function of Hsp40 protein in bacteria

The prototypical Hsp40 protein may be identified as E. coli DnaJ, which was first identified to interact with the Hsp70 protein DnaK and stimulate its ATPase activity; this protein is also implicated in the formation and disassembly of the initiation complex at the origin of λ phage DNA replica-tion in host cells (Yochem et al. 1978; Liberek et al. 1988; Sell et al. 1990). DnaK mainly consists of an N terminal nucleotide-binding domain (NBD; residues 1–366) with ATPase activity and a C terminal substrate-binding domain

(SBD; residues 393–605) with polypeptide binding activ-ity (Bukau and Horwich 1998). DnaK performs folding-assistant functions by interdomain allostery. The NBD can regulate the substrate affinity of the SBD by switching the phosphorylation state between ATP and ADP. DnaK alone exhibits basal ATPase activity (Zylicz and Georgopoulos 1984; Zylicz et al. 1983), whereas the presence of a sub-strate and a DnaJ (Hsp40) protein significantly stimulates DnaK (Hsp70) ATP hydrolysis activity (Tsai and Doug-las 1996). Together with the nucleotide exchange factor Grpe, which stimulates the ADP release rate of DnaK, DnaJ accelerates the cycling of DnaK (Cyr et al. 1994). In vivo assay results have suggested that the DnaK system (DnaK-DnaJ-Grpe) participates in the folding and assem-bly of newly synthesized proteins (Schaffitzel et al. 2001; Deuerling et al. 2003; Genevaux et al. 2004), interacts with unfolded polypeptide chains, prevents their aggregation (Han and Christen 2004), and mediates the disaggregation and refolding of thermolabile proteins (Goloubinoff et al. 1999; Mogk et al. 1999; Zolkiewski 1999). Furthermore, in vitro experiments have shown that the DnaK system can unfold misfolded luciferase, which can refold sponta-neously into a correct configuration after its release from the chaperone system (Schroder et al. 1993; Sharma et al. 2010).

Heat shock protein 40 interact with Hsp70s mainly via the J-domain, specifically the HPD motif. The J-domain alone is sufficient to bind and stimulate the Hsp70 partner (wittung-Stafshede et al. 2003). The substitutions of the HPD motif abolish interactions with partner Hsp70s and ability to stimulate Hsp70 ATPase activity (Mayer et al. 1999; Kluck et al. 2002). A mutant phenotype is manifested when Hsp40 proteins, namely, DnaJ and DjlA, are inacti-vated by Asp-to-Asn substitution in a highly conserved HPD motif; nevertheless, this phenotype can be partially suppressed by introducing DnaKR167H, another DnaK vari-ant, carrying a compensatory mutation at the ATPase region of the NBD domain (Suh et al. 1998; Genevaux et al. 2001).

ZUO1: novel type of Hsp40 protein in fungi

Yeast Hsp40 protein ZUO1 does not belong to any of the three types of E. coli J-protein (Fig. 1), indicating that ZUO1 may emerge in eukaryotes as a representative of a novel Hsp40 subclade. However, ZUO1 still retains an Hsp40 ancestral function similar to bacterial DnaJ. Zuotin efficiently stimulates the ATPase activity of the yeast Hsp70 homolog Ssb1/2 only when ZUO1 forms a complex with another atypical Hsp70 homolog, namely, Ssz1, which may function mainly to facilitate the ability of ZUO1 as an Ssb partner on the ribosome (Huang et al. 2005). Zuotin and

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Ssz1 form a stable heterodimeric ribosome-associated com-plex (RAC); furthermore, RAC, together with Ssb, forms a functional chaperone triad (Gautschi et al. 2001, 2002). Ribosome-associated complex also binds to ribosomes almost quantitatively, and the stoichiometry between Ssz1 and ribosomes is approximately 1:1, whereas the amount of Ssb is twofold to fourfold higher than that of the ribosomes (Gautschi et al. 2001). Ssb can directly bind to short nascent chains as they just exit from the tunnel of ribosomes (Pfund et al. 1998; Hundley et al. 2002; Gautschi et al. 2003). Although RAC fails to come in direct contact with nascent polypeptides, this complex influences the interaction of such polypeptides with Ssb (Gautschi et al. 2001, 2002).

Ribosome-associated complex and Ssb are involved in the regulation of accurate translation termination (transla-tion fidelity) in addition to the widely accepted function in de novo protein folding. The absence of functional RAC or Ssb primarily induces translational read-through (translation termination defect) other than amino acid misincorporation (Rakwalska and Rospert 2004). Chaperone triad-related mutants are sensitive to different factors, including salt, very low temperature, and the aminoglycoside antibiotic paromomycin that binds to the ribosomes near the decod-ing center; these mutants can also interfere with proofread-ing and increase translational error frequency (Luce et al. 1985). Cross-linking approach results (Table 1) revealed that a ribosomal protein of the large subunit Rpl31 is in contact with the ZUO1 subunit of RAC at the polypep-tide tunnel exit. Although Rpl31 is not significant for the anchoring of ZUO1 at the ribosome, genetic evidence has indicated the possibility of both functional couplings (Pei-sker et al. 2008). The depletion of Rpl31 (rpl31a rpl31b) results in slow growth, temperature sensitivity, paromo-mycin hypersensitivity, and defects in translational fidel-ity, and these observations are consistent with RAC-related mutation (Peisker et al. 2008). In addition to a highly con-served J-domain, other domain organization characteristics are observed in ZUO1; for instance, a ZUO1 homologous region found in all eukaryotic ZUO1 orthologs is local-ized at the N terminus; a highly charged region (amino acids 285–364, Fig. 1b) adjacent to the C terminus, and this region is necessary to anchor ZUO1 at the ribosome and permit ZUO1 interaction with nucleic acids (Yan et al. 1998). A deletion version of ZUO1 lacking amino acids at positions 281–331 can still interact with Ssz to form a com-plex, but the complex fails to attach to the ribosome (Peisker et al. 2008). Besides widely accepted function in cytosol, ZUO1 has critical roles in nucleus. For instance, ZUO1 and Ssb mediate nuclear steps of ribosome biogenesis, includ-ing rRNA maturation, ribosomal export from the nucleus, and the recycling of the shuttling factor Arx1 as a nuclear export receptor of the 60S subunit (Albanese et al. 2010). Zuotin and Ssb participate in the maturation of 35S rRNA at

the beginning of ribosome biogenesis. Furthermore, ZUO1 associates with nuclear 60S ribosomal intermediates, medi-ating the processing of the 27S rRNA precursor and subse-quently the formation of the mature 25S rRNA. Zuotin is predominantly localized in the cytosol but also shuttles between the cytoplasm and the nucleus. Zuotin devoid of the charged C terminal region (ZUO1-ΔC) is mostly local-ized in the nucleolus (Albanese et al. 2010), indicating that the C terminus may contain the nuclear export signal (NeS) in ZUO1. Similarly, Ssb containing a functional leucine-rich NeS in the C terminus similarly shuttles between the cyto-plasm and the nucleus (Shulga et al. 1999). Another direct example of ZUO1 function in the nucleus is implicated in transcriptional regulation. ZUO1 can interact with the Zn2-Cys6 cluster transcription factor Pleiotropic drug resistance 1 (Pdr1) via 69 residues (amino acids at positions 365–433) in the C terminus; as a result, Pdr1 is activated, pleiotropic drug resistance is induced, and premature cell growth arrest occurs during diauxic shift (Prunuske et al. 2012; Ducett et al. 2013). The C terminal domain (CTD) of ZUO1 con-sisting of 86 residues (amino acids at positions 348–433) can fold automatically into inhibitory four-helix bundle where the hydrophobic residues required for Pdr1 binding and activation are sequestered. By contrast, the unfolding of the CTD domain causes ZUO1 to dissociate from the ribo-some, prevents auto-inhibition, exposes critical hydrophobic residues, and activates Pdr1 (Ducett et al. 2013). Therefore, the dual effect of CTD domain in ZUO1 via conformation alteration bridges the communication between the cytosolic translation and the nuclear transcription, suitable for induc-ing a rapid response to dynamic environmental conditions.

High-throughput protein interactome strategies cou-pled with affinity purification and mass spectrometry in yeast provide as many candidate partners as possible for a specific target protein (Gavin et al. 2006; Guerrero et al. 2008). Current information on ZUO1 as prey has revealed approximately 50 potential partners (Table 1). Some part-ners have been well identified to participate in specific events, such as protein folding (Ssb2 and Ssz1) and ribo-some biogenesis (ARB1, NMD3, eCM1, and NSA3). Other partners are mainly involved in ribosome binding (RPLs, RPSs, and ASC1), translation initiation (TIF4631 and HYP2) and elongation (eFB1), proteasomal biosynthe-sis (RPN10, RPN11, and BLM10) and protein degradation (CIC1, TOM1, and UBI4), epigenetic regulation (SMT3 and IeS1), cell cycle (AMN1), bud site selection (BUD14), and other unclassified functions.

ZUO1 orthologs in animals

Studies on ZUO1-related factors (ZRFs) in animals have focused on nematodes, mice, and humans. In

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Table 1 Both DnaJ- and ZUO1/ZRF1-associated partners identified mainly via high-throughput methods

Gene Partner Partner function Method Reference

Escherichia coli

DnaJ DnaK* Hsp70 protein, molecular chaperone SPR Mayer et al. (1999)

YajL Oxidative stress resistance chaperone A-MS Le et al. (2012)

Saccharomyces cerevisiae

ZUO1 Ssz1* Polypeptide folding A-MS/ Fiaux et al. (2009)

A-w Conz et al. (2007)

Ssb* Polypeptide folding A-MS/ Huang et al. (2005)

A-w Krogan et al. (2006)

GIS2 Translational activator for mRNAs with internal ribosome entry sites

A-MS Sammons et al. (2011)

TIF4631 Translation initiation; biogenesis of the large ribosomal subunit

A-MS Gavin et al. (2002, 2006)

HYP2 Translation initiation A-MS Jao and Chen (2006)

eFB1 Translation elongation A-MS Collins et al. (2007)

BLM10 Proteasome activator subunit A-MS Krogan et al. (2006)

RPN10 Non-ATPase base subunit of the 19S regulatory particle of the 26S proteasome

A-MS Guerrero et al. (2008)

RPN11 Metalloprotease subunit of the 19S regulatory particle of the 26S proteasome lid

A-MS Guerrero et al. (2008), Kaake et al. (2010)

TOM1 e3 ubiquitin ligase of the hect-domain class A-MS Krogan et al. (2006)

UBI4 Ubiquitin marker for degradation via proteasome system A-MS Ziv et al. (2011)

ARB1* ABC family, involved in 40S and 60S ribosome biogenesis A-MS/A-w Dong et al. (2005)

CIC1/NSA3* Biogenesis factor for 66S pre-ribosome A-w Albanese et al. (2010)

eCM1* Nuclear export of the 60S ribosomal subunit A-w Albanese et al. (2010)

NMD3* Nuclear export of the 60S ribosomal subunit A-w Albanese et al. (2010)

SGD1 Biogenesis of 40S ribosomal subunit A-MS Krogan et al. (2006)

RPL31a/b* Component of 60S ribosomal subunit cross-linking Yan et al. (1998), Peisker et al. (2008)

RPL24a/b* Component of 60S ribosomal subunit A-w Albanese et al. (2010)

RPL3, 12a, 25, 27a, 30, 31a/b

Component of 60S ribosomal subunit A-MS Gavin et al. (2006)

RPS3, 4a, 5, 7a, 11a, 13, 21a, ASC1

Component of 40S ribosomal subunit A-MS Gavin et al. (2002, 2006)

AMN1 Daughter cell separation, multiple mitotic checkpoints, and chromosome stability

A-MS Gavin et al. (2002, 2006)

BUD14 Involved in bud site selection A-MS Gavin et al. (2002)

LYS4 Homoaconitase, involved in the lysine biosynthesis pathway A-MS Krogan et al. (2006)

TGL5 Bifunctional enzyme with TAG lipase and LPA acyltransferase activity

A-MS Krogan et al. (2006)

MIS1 Mitochondrial C1-tetrahydrofolate synthase A-MS Gavin et al. (2002, 2006)

GPD2 NAD-dependent glycerol 3-phosphate dehydrogenase A-MS Krogan et al. (2006)

SMT3 SUMO family; regulating chromatid cohesion and chromosome segregation

A-MS Felberbaum et al. (2012)

IeS1 Subunit of the INO80 chromatin remodeling complex A-MS Krogan et al. (2006)

MeT4 Leucine-zipper TF, regulating the sulfur amino acid pathway Y2H Newman et al. (2000)

SOK1 Involved in cAMP-mediated signaling A-MS Krogan et al. (2006)

PDR1* Zinc cluster TF, activating multidrug resistance genes Y2H Prunuske et al. (2012), Ducett et al. (2013)

YPT6 Ras-like GTP binding protein involved in the secretory pathway

A-MS Krogan et al. (2006)

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Caenorhabditis elegans, ZUO1/ZRF ortholog DNJ11 is implicated in asymmetric cell division and subsequent apoptosis (Hatzold and Conradt 2008). The DNJ11-CeS2-CeS1 three-gene pathway mediates the asymmetric divi-sion of the neuroblast by regulating the displacement of the mitotic spindle and the orientation of cell division axis, consequently giving rise to the neurosecretory moto-neuron (NSM) and NSM sister cell (Hatzold and Conradt 2008). Moreover, DNJ11 cooperates with CeS2 to sup-press and exclude CeS1 expression from the NSM sister cell, consequently causing the apoptosis of the NSM sister cell; J-domain and SANT domain are necessary for DNJ11 activity (Hatzold and Conradt 2008). The dnj-11 mutant facilitates the survival of NSM sister cells. However, the mutant also displays other morphological defects in embry-onic, larval, and vulva development, as well as embryonic lethality, slow growth, and decreased brood size (Hatzold and Conradt 2008). DNJ11 exhibits an almost ubiquitous expression pattern and cytoplastic localization detected by the DNJ11p::DNJ11:GFP reporter (Hatzold and Conradt 2008).

The mouse ZUO1 ortholog MIDA1 (Mouse Id Associ-ated 1) interacts with the HLH region of inhibitor of dif-ferentiation (Id) via the N terminal conserved region M (amino acids at positions 188–256, Fig. 1b) and inhib-its the activity of Id, thereby promoting cell growth and inhibiting the differentiation of murine erythroleukemia (MeL) cells (Shoji et al. 1995). Mouse Id Associated 1 exhibits two distinct DNA-binding activities: a Z-DNA-binding activity near the N terminal ZUO1 domain simi-lar to the yeast ortholog ZUO1 (Zhang et al. 1992) and a

sequence-specific DNA-binding activity near the C termi-nal SANT repeats. However, Id interaction with MIDA1 can stimulate sequence-specific DNA-binding activity and interrupt Z-DNA-binding activity. Thus, MIDA1 may mediate the function of Id in growth promotion by switch-ing DNA-binding activities (Inoue et al. 1999, 2000). RNA blot results have shown that MIDA1 is expressed in undif-ferentiated MeL cells at a higher extent than in differen-tiated cells. Under normal conditions, the highest level is observed in the testes and in the spleen; by contrast, no signal is detected in other examined tissues, such as brain, heart, lung, muscle, kidney, and liver (Shoji et al. 1995).

The human ZUO1 ortholog ZRF1/MPP11/DNAJC2 was first identified as an M phase phosphoprotein involved in mitotic division (Matsumoto-Taniura et al. 1996). MPP11/ZRF1 also inherits the ancestral function, in which this ortholog, together with atypical Hsp70 homolog Hsp70L1, interacts with ribosomes and forms mam-malian ribosome-associated complex (mRAC); mRAC can partly complement the yeast homologous complex RAC mutant for both cold and paromomycin sensitiv-ity (Otto et al. 2005). Furthermore, MPP11/ZRF1 knock-down causes slow growth and hypersensitivity to amino-glycoside G418 (Jaiswal et al. 2011), in accordance with a ZUO1/ZRF homolog defect in examined species. The MPP11/ZRF1 truncated version that lacks both SANT domains retains partial activity in yeast; this result sug-gests that the ribosomal function of MPP11/ZRF1 may not require SANT domains. The Hsp70L1 level was reduced in the MPP11/ZRF1-knockdown cell lines, indicating that MPP11/ZRF1 can enhance Hsp70L1 stabilization (Jaiswal

Table 1 continued

Gene Partner Partner function Method Reference

SKI3 The degradation of aberrant mRNAs A-MS Gavin et al. (2002, 2006), Collins et al. (2007)

SCP160 RNA-binding protein, involved in translating A-MS Gavin et al. (2002, 2006), Collins et al. (2007)

KIN3 Ser/Thr protein kinase A-MS Krogan et al. (2006)

Caenorhabditis elegans

Dnj11 Y17G9B.5 Unknown Y2H Li et al. (2004)

Homo sapiens

ZRF1 Hsp70L1/HSPA14* Translational fidelity Otto et al. (2005)

H2Aub* Transcriptional repression marker A-MS Richly et al. (2010)

Mus musculus/Rattus norvegicus

MIDA1 Id* Cell differentiation A-w Shoji et al. (1995)

PLCβ1 Regulation of cell division and the balance between cell proliferation and differentiation

A-MS Piazzi et al. (2013)

Volvox carteri

GlsA Hsp70A* Asymmetric cell division A-w Cheng et al. (2005)

A-MS affinity capture-MS, A-W affinity capture-western, SPR surface plasmon resonance, Y2H yeast two hybrid

* The interaction confirmed by other methods

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et al. 2011). MPP11/ZRF1 functions as a histone modifi-cation reader in the nucleus in addition to its functions in translational fidelity control in cytosolic ribosomes. Pull-down assay is performed to detect the binding capacity of truncated MPP11/ZRF1 versions with GST-ubiquitin; the results show that the UBD is adjacent to the C termi-nus of the J-domain, mostly overlapping with the M region of the mouse MIDA1 (Fig. 1b) (Richly et al. 2010). The SANT domain is commonly associated with chromatin remodeling factors but not required for ubiquitin bind-ing in MPP11/ZRF1; by comparison, SANT2 domain is required for histone binding in the green alga ortholog GlsA (Pappas and Miller 2009). ZRF1/MPP11 can com-pete with RING1B for H2AK119ub binding and displace RING1B-containing polycomb repression complex 1 (PRC1) from chromatin during retinoic acid-induced neu-ronal differentiation of the human teratocarcinoma cell line. Yeast ZUO1 also exhibits weak ubiquitin-binding activity but fails to be recruited to chromatin and displace PRC1 (Richly et al. 2010). In vitro experiments have fur-ther shown that ZRF1/MPP11 can facilitate the removal of H2Aub by recruiting the specific deubiquitinase USP21 in an unknown mechanism; as a result, the transcription of polycomb target genes is activated (Richly et al. 2010). Considering that both Id and H2Aub bind to a similar M domain/UBD region of MIDA1/ZRF1, Richly and Di Croce (2011) revealed that Id-ZRF1 interactions may retain ZRF1 in the cytoplasm and block ZRF1 recruitment to chromatin marked with H2Aub; thus, differentiation is inhibited. As expected, Id can enhance RING1b e3 ligase activity, leading to H2AK119ub accumulation in target genes (Qian et al. 2010). MPP11/ZRF1 is also implicated in senescence and tumorigenesis. The INK4-ARF locus is epigenetically silenced by polycomb proteins in healthy cells (Ribeiro et al. 2012), but certain stimuli, such as onco-gene misexpression, may reactivate the INK4-ARF locus that encodes three related proteins, namely, p15INK4b, ARF, and p16INK4a, and induce these proteins to function as tumor suppressors and synergistically initiate senescence response. During oncogene-induced senescence, ZRF1 expression is enhanced, and ZRF1 can be directly recruited to the promoter regions of the INK4-ARF locus and acti-vate transcription; as a result, PRC1-mediated silencing is antagonized at this locus. By contrast, the depletion of ZRF1 in oncogenic Ras-expressing cells leads to the reduced expression of p16INK4a and ARF; this reduced expression bypasses cellular senescence but restores prolif-eration and facilitates tumorigenesis (Ribeiro et al. 2012). Chromosomal aberrations involving MPP11/ZRF1 are linked to primary head and neck squamous cell tumors (Resto et al. 2000). As such, MPP11/ZRF1 can be used as important candidates for cancer detection and treatment (Resto et al. 2000; Greiner et al. 2003, 2004). In healthy

tissues, MPP11/ZRF1 is specifically and strongly expressed in the testes (Greiner et al. 2003). However, MPP11/ZRF1 is extensively upregulated in leukemic blasts and consid-ered as an attractive target for leukemia T cell therapy (Al Qudaihi et al. 2010).

ZUO1/ZRF orthologs in plants

The spherical multicellular green alga Volvox carteri com-prises only two cell types: small biflagellate somatic cells and large immortal germ cells (Miller and Kirk 1999); this alga still exhibits a complete germ-soma division of labor (Kirk 2001). The Volvox ZUO1/ZRF ortholog Gonidialess A (GlsA) is required for asymmetric divisions, which are necessary to establish germ and somatic cell fate during embryogenesis. glsA belongs to one of the transposon-inserted gls mutants, which only undergo symmetric cell divisions that induce the differentiation of only small somatic cells. These gls mutations can be recovered and maintained in the genetic background of somatic regen-erator (regA) mutation, which permits somatic cells to change their developmental fate into a germ cell and subsequently induce gonidium formation (Kirk et al. 1999). Gonidialess A is localized in the nucleus and in a small cytoplasmic region around nucleus during inter-phase; GlsA is also localized in the spindle region dur-ing mitosis (Miller and Kirk 1999; Cheng et al. 2005), which is consistent with its function in asymmetric divi-sion. Retaining the ancestral role, GlsA can interact with the predicted chaperone Hsp70A. The co-expression of HA-tagged HA-GlsAD147N and Hsp70AR175H-HA variants partially rescues a glsA mutant phenotype, mimicking the partial activity recovery exhibited by the co-existence of DnaJD35N and DnaKR167H with similar mutated sites (Suh et al. 1998; Genevaux et al. 2001). The results of immu-nofluorescence analysis using a specific antibody have shown that GlsA is evenly distributed in all of the exam-ined cleavage-stage blastomeres; by comparison, Hsp70A is more abundant in anterior blastomeres (for asymmetri-cal division) than in posterior ones (for symmetrical divi-sion). These results indicate that Hsp70A may determine the locations of asymmetric divisions and germ-soma dif-ferentiation in Hsp70A-GlsA chaperone partners (Cheng et al. 2005). The glsA mutant exhibits cold sensitivity and paromomycin ultrasensitivity; these characteristics are similar to those observed in yeast ZUO1 and human MPP11/ZRF1 that function as ribosome-associated fac-tors regulating translation fidelity (Pappas and Miller 2009). The SANT2-lacking variant completely restores the paromomycin hypersensitivity of the glsA mutant but fails to recover asymmetric division defect; this result indicates that GlsA may perform separate functions in

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both aspects. In addition to SANT2 domain, J-domain and M domain (corresponding to UBD of human ZRF1) are critical for GlsA function in asymmetric division (Miller and Kirk 1999; Pappas and Miller 2009). The SANT2 domain and the M domain (to a lesser extent) are required for GlsA binding to histones, but J-domain is not required (Pappas and Miller 2009).

In higher plants, very limited information has been reported regarding a ZUO1/ZRF ortholog in Lilium longiflorum, namely, LlGlsA. Semi-quantitative RT-PCR has revealed that LlGlsA is ubiquitously expressed in

examined organs manifesting a weak signal in the leaf, root, stem, style, ovule, microspore and early bicellular, and by comparison, a strong signal in late bicellular pol-len and generative cell (Mori et al. 2003). The immuno-fluorescence analysis results of anti-LlGlsA have further confirmed the strongest expression in the generative cell at the late bicellular pollen stage (Mori et al. 2003), but this result differs from the GlsA transcriptional peak in gonidium formation during asymmetric divisions, indicat-ing the functional divergence of ZUO1/ZRF orthologs in plant evolution.

Fig. 2 Phylogenetic construc-tion of ZUO1/ZRF orthologs mainly in green lineage. a Phylogeny of ZUO1/ZRF orthologs; ZUO1-like proteins used in this study were identi-fied by protein BLAST using ZRF1, ZUO1, and AT5G06110 (atDjC2/AtZRF1b) as que-ries against different protein sequence database: NCBI (http://blast.ncbi.nlm.nih.gov), phytozome (http://www.phytozome.net). Approximately, 60 ZUO1-like proteins were aligned using Clustalw (Thompson et al. 1994), and phylogenetic analysis was performed using MeGA5.1 (Tamura et al. 2007). Closed diamond DnaJ, closed triangles reported ZRF in meta-zoan, closed circle ZUO1, anti-triangle GlsA, open squares, Arabidopsis ZRF. b Gain or loss of SANT domain in some plant ZUO1/ZRF proteins. Only well-established domains are shown in this figure. J J-domain, S SANT domain. OsZRF1, Oryza sativa LOC_Os04g30890

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Phylogenic analysis of ZUO1/ZRF proteins in green lineage

According to the classification based on domain organi-zation, ZUO1/ZRF proteins can be classified as type III Hsp40s. However, true ZUO1/ZRF orthologs cannot be clearly assigned in prokaryotes when phylogenetic analy-sis is also performed (Fig. 1a). Zuotin/Zuotin-related fac-tor proteins may represent a novel type of Hsp40 subfamily specific to eukaryotes. To investigate the evolutionary con-servation of ZUO1/ZRF orthologs in green lineage, we per-formed phylogenetic analysis using maximum likelihood (ML) methods in MeGA5.1 package (Fig. 2a). In addition to ZUO1-domain, a pair of SANT domains is present at the C terminus of ZUO1/ZRF orthologs in animals and plants but not in fungi. Almost all eukaryotic organisms with openly published genome sequences only have a single copy of the ZUO1/ZRF proteins. Intriguingly, three copies are found in the moss Physcomitrella patens (PpZRF11-13). In angiosperms, monocot ZUO1/ZRF orthologs from Poaceae and Liliaceae typically show a single copy, but a limited number of exceptions (two copies) are observed in dicot plants, such as cotton GrZRF11/12, soybean GmZRF11/12, poplar PtZRF11/12, and most of Brassi-caceae orthologs; even Brassica rapa contains four copies. Therefore, gene duplication events may occur twice in the moss Physcomitrella patens, at least once with the forma-tion of Brassicaceae and one more time in B. rapa. The gain or loss of the SANT domain occasionally occurs in certain plant ZUO1/ZRF proteins (Fig. 2b). For example, the green alga MpZRF1 (Xp003062045) only harbors a single SANT domain, whereas the common monkey flower MgZRF1 (Mgv1a021454m) contains three SANT domains. In B.

rapa, BrZRF11 (Bra034841) and BrZRF14 (Bra001404) contain normal double SANT domains, whereas both BrZRF13 (Bra005875) and BrZRF12 (Bra005877) are short of the SANT domain, which is reminiscent of the yeast ZUO1. Oryza sativa OsZRF1 (Os04g30890), which lacks the key J-domain and is excluded from a recent rice J-proteins classification (Sarkar et al. 2013), consists of only two SANT domains compared with its distant relative Oryza brachyantha ObZRF1 (XP_006652199) with a nor-mal domain architecture (Fig. 2b). This result indicates that the ZRF1-like protein of cultivated rice may result from the loss of the J-domain of the wild ancestor of rice during domestication. Therefore, multiple strategies are applied during the evolution of ZUO1/ZRF orthologs.

The number of introns and the length of ZUO1/ZRF genes vary in different kinds of eukaryotes (Fig. 3). In plants, zero to only one intron is found in higher plants (embryophyta), but a maximum of 16 introns have been found in green algae (Chlorophyta), indicating that higher plant ZRF genes have lost almost all introns during evolu-tion. Fungus ZRF normally contains 0–6 introns. In ani-mals (Metazoa), 15–16 introns are found in higher animals (vertebrate), such as mammals, birds, reptiles, amphib-ians, and fishes (Fig. 3) compared with typical 0–2 introns in insects (data not shown). Choanoflagellata, as the clos-est related unicellular protist group to Metazoa (Lang et al. 2002; King et al. 2008; Fairclough et al. 2013), have approximately 16 introns in ZRF genes similar to those found in higher animals, but these introns are extremely extended (Fig. 3): for instance, human MPP11 introns cover approximately 15/16 full-length genomic sequence. These results suggest that ZRF genes in higher animals maintain the ancestral gene structure except overlong

Fig. 3 exon–intron pattern of several representative ZUO1/ZRF genes. The genomic DNA (gDNA) and cDNA schematic of human HsZRF1, mouse MIDA1, Danio rerio DrZRF1, Drosophila

DmZRF1, nematode DNJ11, Salpingoeca rosetta SrZRF1, Vovlox GlsA, Arabidopsis AtZRF1a, and yeast ZUO1 are shown. Red/pink rectangle exon, straight line intron. Scale bar 1 kb

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introns, but exon–intron pattern formation in other Meta-zoa may have resulted from intron deletion and exon com-bination. Overall, the exon–intron structure of ZUO1/ZRF gene possibly evolves toward opposite directions in higher animal and plant species. The ratio of ZUO1/ZRF intron length to gene length (intron/gene) is remarkably increased in higher animal branch; by contrast, the majority of higher plants is devoid of introns and composed of a single large exon. Considering the extremely opposite behavioral char-acteristics between higher animals and higher plants, we speculated that the proportion of introns in the ZUO1/ZRF gene may be positively related to the behavioral complex-ity of organisms. The genes with a higher intron/gene ratio may permit the accession of more transcriptional factors and subsequently facilitate more accurate regulation; thus, a more complicated behavior is achieved.

J-domain of higher plant ZUO1/ZRF ortholog display the highest conservation

To evaluate the conservation of the J-domains of ZUO1/ZRF proteins throughout evolution, we identified and analyzed these domains from different species via online SMART database and Clustalw program. we ini-tially analyzed and then obtained each consensus sequence of J-domain within ZUO1/ZRF orthologs from fungi, Metazoa, green algae, higher plants, and two phyla in Pro-tista (Fig. 4a; Online Resources 2, 3, 4, 5, 6, 7). Choano-zoa and Ameobozoa were selected to represent the mixed population of protists because this kingdom consists of a diverse group of single-celled eukaryotes, which are typi-cally not closely related to one another (Online Resource 1). Amino acid alignment showed that J-domains consist of 60–70 conserved residues in fungi, Ameobozoa, Choa-nozoa, Metazoa, and green algae, and even 76 conserved residues in higher plants (Fig. 4a). J-domain consensus sequence showed the highest amino acid conservation (49 % identity) in higher plants compared with ~38 % identity in fungi, ~34 % identity in Ameobozoa, ~36 % identity in Choanozoa, ~29 % identity in green algae, and ~21 % identity in Metazoa. The J-domain in Choanozoa displays almost the same length and great similarity to that in multicellular Metazoa; this result is consistent with the commonly accepted viewpoint that both phyla belong to the closest sister clades in evolutionary relationship (Shalchian-Tabrizi et al. 2008). However, the J-domain of Choanozoa is distinguished from unicellular Ameobozoa classified under the same kingdom (Protista). This result also indicates that Protista could be considered as a poly-phyletic group. The HPD motif and subsequent K resi-due (HPDK) are highly conserved in all of the examined ZUO1/ZRF orthologs, whereas K residue is not conserved

in prototypical Hsp40s in prokaryotes (Fig. 4a). Bacte-rial DnaJ and its potential orthologs YDJ1 in yeast and HDJ2 in human have one to two gaps compared with the J-domain in eukaryotic ZUO1/ZRF proteins. In eukary-otes, gap I-filled region is localized between helices I and II and normally contains three residues; the central resi-due R is highly conserved. Gap II-filled region is the most prominent in higher plants and predicted to form another helix structure (Fig. 4a). These results suggest that at least

Fig. 4 Domain analysis of ZUO1/ZRF protein. a Multiple align-ment of the J-domain within the ZUO1/ZRF protein. The coloring scheme indicates conserved residues: red > pink > green. X repre-sents any residues. The dash line between two question marks indi-cates uncertain residue deletion or insertion. The dash line indicates deleted residues. Conserved hydrophobic residues are indicated by @ above the alignment. Consensus secondary structure of J-domain is shown below the alignment. Alpha-helices and beta-strands are indi-cated by green helices and blue arrows, respectively. each sequence above the dotted line is the predicted J-domain consensus sequence of the ZUO1/ZRF protein derived from fungi, metazoans, green algae, higher plant, and two phyla of protists, respectively (Online Resource 2, 3, 4, 5, 6, 7). The highlighted colors in each line represent the rela-tive conservation in each type of organisms but not compared with the others. The J-domain sequences below the dotted line originate from human HDJ2, yeast YDJ1, and E. coli DNAJ belonging to the type I Hsp40 proteins, type II Hsp40 CbpA in E. coli, and type III Hsp40 DjiA in E. coli. The highlighted colors represent the relative conservation among the selected sequences. b Representative mul-tiple alignments of ZUO1 domains within ZUO1/ZRF proteins. The ZRF orthologs for the analysis of ZUO1 domains in this figure are listed as follows: yeast ZUO1; human HsZRF1; Arabidopsis AtZRF1a/b; lily LlGlsA; rice OsZRF1; and Volvox vcGlsA. High-lighted color in cyan indicates J-domain. Highlighted color in yel-low indicates the mutated sites (SwReF→AvAvA) disrupting the interaction with histone in Volvox GlsA. The differently colored let-ters indicate relative conservation (red > blue > green). The under-line sequences in HsZRF1 and MIDA1 indicate UBD domain and M region, respectively. The number at each end of the sequence indi-cates the residue numbers. c Multiple sequence alignments of both SANT domains within ZUO1/ZRF orthologs. The coloring scheme indicates conserved residues: red > pink > green. X indicates any residues. The dash line indicates deleted residues. Conserved hydro-phobic residues are indicated by @ above the alignment. Underlines highlight the bulky hydrophobic residues that are predicted to form the hydrophobic core of the SANT domain. Consensus secondary structure of SANT domain is shown below the alignment. Alpha-hel-ices are indicated by green helices. The number labeled on the right indicates the corresponding residue numbers. each sequence in the first block is the calculated SANT1- and SANT2-domain consensus sequences derived from metazoans, higher plants, and Amoebozoa, respectively (Online Resource 2, 5, 7). The highlighted colors in each line represent the relative conservation in each type of organisms but not compared with other organisms. Considering the very small sam-ple size and less conservativeness of SANT domains in green algae and Choanozoa as the most closely related unicellular eukaryotes of higher plants and metazoans, respectively, we only showed the con-sensus sequences of both SANT domains within ZUO1/ZRF1 pro-teins from higher plants, metazoans, and Amoebozoa. The middle block indicates the classical SANT domains from human SMRT and yeast Ada2. The highlighted colors in the middle and last blocks rep-resent the relative conservation among the selected sequences

▸

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one or two insertion events occur during the formation of ZUO1/ZRF clades in specific eukaryotes; thus, sequence divergence and novel gene generation occur. In contrast to

bacterial J-proteins, J-domains in eukaryotic ZUO1/ZRF proteins are usually embeded in larger ZUO1-domains (Fig. 4b), which also contain a UBD domain/M region.

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Both SANT domains may function as a complete module in ZRFs

The SANT domain can function as a unique histone modi-fication reader in chromatin remodeling by coupling histone binding with enzyme catalysis (Boyer et al. 2004). In numer-ous Gcn5-containing histone-acetyltransferase (HAT) com-plexes, the defective subunit Ada2 with a small deletion in the SANT domain causes HAT inactivity in the whole com-plex and reduces the binding ability to unacetylated histone H3 tails (Boyer et al. 2002; Barbaric et al. 2003). In the his-tone deacetylase (HDAC) complex SMRT/N-CoR/HDAC3, SMRT and N-CoR contain a pair of SANT domains and are required for the deacetylase activity of the catalytic subunit HDAC3 (Guenther et al. 2001; Yu et al. 2003). SANT1 and SANT2 of SMRT perform distinct and mutually irreplace-able functions, accounting for the interaction of SMRT with HDAC3 and the binding of SMRT to the histone tail, respec-tively (Guenther et al. 2001; Yu et al. 2003). Both SANT domains likely promote and maintain histone deacetylation synergistically (Boyer et al. 2004). In this study, almost all eurkaryotic ZRF-like proteins contain a pair of C terminal SANT domains except fungal ZUO1 proteins, which may have evolved from ZRF1-like factors by lineage-specific loss of SANT domains (Braun and Grotewold 2001). In addi-tion, SANT domain is possibly specific for eukaryotes as indicated by the absence of this domain in the E. coli gene database (http://www.ecogene.org; Zhou and Rudd 2013) via BLAST search using SANT fragments as queries. Phyloge-netic analysis based only on SANT domains revealed that both SANT domains of ZRF proteins are grouped together to form two sister clades (S1 and S2); as such, these domains are separated from classical SANT domains in SMRT or Ada2 and Myb DNA-binding repeats in an MYB protein (Online Resource 8). This result suggests that both SANT1 and SANT2 may co-evolve as an intact functional unit within ZRF proteins. Although one single SANT-containing ZRF proteins have been identified, other SANT-like regions can still be traced. For instance, Micromonas pusilla MpZRF1 contains a well-identified SANT2 domain, together with an abnormal SANT1-like region with high e-value (Online Resource 3). Salpingoeca rosetta SrZRF1 consists of a well-verified SANT1 domain and an incomplete SANT2 domain with premature termination (Online Resource 6). The species cluster in the SANT2 clade is consistent with well-established classifications of animals and plants, whereas SANT1 clade remains a little confused (Online Resource 8); this variation indicates that SANT2 may be more conserved than SANT1. Sequence alignments further showed that the core bulky aro-matic residues at the first two α-helices are strictly conserved in both SANTs, but the core one at the third α-helix is only relatively stable in SANT2 and is substituted mostly by A, T, or v residues in SANT1 (Fig. 4c); this structure corresponds

to a more conserved function of SANT2 than SANT1. There-fore, both SANT domains should undergo co-evolution and function as one unit in ZRF-like proteins, in which SANT2 is more conserved than SANT1.

Conclusion

Heat shock protein 40 prototypical protein DnaJ mainly functions as the co-chaperone of the molecular chaperone DnaK (Hsp70 protein) for the folding of nascent polypep-tides. eukaryotic ZUO1/ZRF proteins have also inherited this ancestral function; for instance, yeast ZUO1, human ZRF1, and Volvox GlsA can bind to their correspond-ing Hsp70 chaperones. In addition, ZUO1/ZRF proteins acquired certain neofunctions as novel domains have been gained and more complicated lifestyle has emerged. For instance, firstly, ZUO1/ZRF proteins participate in main-taining translational fidelity. Disruption in yeast ZUO1, human MPP11, and green alga GlsA causes both cold and aminoglycoside sensitivity. Secondly, ZUO1/ZRF proteins are implicated in asymmetric division and polar establishment. The yeast ZUO1 is involved in determin-ing the polar selection of a bud site (Ni and Snyder 2001; Pappas and Miller 2009). The nematode DNJ11 mediates the asymmetric division of NSM neuroblast. The green alga GlsA is necessary to establish germ and somatic cell initials during embryogenesis. The lily LlGlsA may be involved in the differentiation of generative cells in the developing pollen. Thirdly, ZUO1/ZRF proteins are involved in mitosis and cell cycle. MPP11/ZRF1 was ini-tially identified as an M phase phosphoprotein. The cyto-metric analysis and the BrdU incorporation assay revealed that the reduction of mouse MIDA1 blocks the progres-sion of DNA synthesis during cell cycle (Shoji et al. 1995). Fourthly, ZUO1/ZRF proteins also have distinct functions in the nucleus. The yeast ZUO1 protein medi-ates several nuclear steps of ribosome biogenesis and tran-scriptional regulation of Pdr1. Human MPP11/ZRF1 can replace PRC1 and bind to the H2Aub epigenetic marker via the UBD domain. Hence, ZUO1/ZRF proteins not only assist protein folding and maturation, control trans-lation fidelity, or function as a signal component in cyto-plasm, but also assist ribosomal biogenesis and function as a transcriptional factor or chromatin remodeler in the nucleus. Identification and classification of ZUO1/ZRF orthologs, especially in higher plants, provide a platform for future investigation on the biological function of this important family of proteins.

Acknowledgments This work was supported by Research Founda-tion of Hunan Provincial education Department of China (13B045) and National Basic Research Program of China (973 Program, 2012CB910500).

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Conflict of interest The authors declare they have no conflict of interest.

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