Plant Mol Biol (2014) 85:443–458 DOI 10.1007/s11103-014-0196-7

Divergence of the expression and subcellular localization of CCR4-associated factor 1 (CAF1) deadenylase in Oryza sativa

Wei-Lun Chou · Li-Fen Huang · Jhen-Cheng Fang · Ching-Hui Yeh · Chwan-Yang Hong · Shaw-Jye Wu · Chung-An Lu

Received: 18 November 2013 / Accepted: 25 April 2014 / Published online: 8 May 2014 © Springer Science Business Media Dordrecht 2014 +

Abstract Deadenylation, also called poly(A) tail shorten- tissues. OsCAF1B was transient, induced by drought, cold, ing, is the first, rate-limiting step in the general cytoplasmic abscisic acid, and wounding treatments. OsCAF1H mRNA mRNA degradation in eukaryotic cells. The CCR4-NOT was not detected either under normal conditions or dur- complex, containing the two key components carbon cat- ing most stress treatments, but only accumulated during abolite repressor 4 (CCR4) and CCR4-associated factor 1 heat stress. Four OsCAF1-reporter fusion proteins were (CAF1), is a major player in deadenylation. CAF1 belongs localized in both the cytoplasm and nucleus. In addition, to the RNase D group in the DEDD superfamily, and is a when green fluorescent fused with OsCAF1B, protein conserved through evolution from yeast to humans OsCAF1G, and OsCAF1H, respectively, fluorescent spots and plants. Every higher plant, including Arabidopsis and were observed in the nucleolus. OsCAF1B fluorescent rice, contains a CAF1 multigene family. In this study, we fusion proteins were located in discrete cytoplasmic foci identified and cloned four OsCAF1 (OsCAF1A, and fibers. We present evidences that OsCAF1B colocalizes OsCAF1B, OsCAF1G, and OsCAF1H) from rice. Four with AtXRN4, a processing body marker, and AtKSS12, a recombinant OsCAF1 proteins, rOsCAF1A, rOsCAF1B, microtubules maker, indicating that OsCAF1B is a compo- rOsCAF1G, and rOsCAF1H, all exhibited 3′–5′ exonu- nent of the plant P-body and associate with microtubules. clease activity in vitro. Point mutations in the catalytic Our findings provide biochemical evidence that OsCAF1 residues of each analyzed recombinant OsCAF1 proteins proteins may be involved in the deadenylation in rice. The were shown to disrupt deadenylase activity. OsCAF1A and unique expression patterns of each OsCAF1 were observed OsCAF1G mRNA were found to be abundant in the leaves in various tissues when undergoing abiotic stress treat- of mature plants. Two types of OsCAF1B mRNA transcript ments, implying that each CAF1 in rice plays a spe- were detected in an inverse expression pattern in various cific role in the development and stress response of a plant.

Keywords CCR4-associated factor 1 (CAF1) · Electronic supplementary material The online version of this Deadenylase · Stress response · Subcellular localization · article (doi:10.1007/s11103-014-0196-7) contains supplementary Processing body · Oryza sativa material, which is available to authorized users.

W.-L. Chou · J.-C. Fang · C.-H. Yeh · S.-J. Wu · C.-A. Lu (*) Department of Life Sciences, National Central University, Introduction Jhongli City, Taoyuan County 320, Taiwan, ROC e-mail: [email protected] Higher land plants are sessile and require complex yet L.-F. Huang coordinated gene activities to withstand various stresses. Graduate School of Biotechnology and Bioengineering, Yuan Ze in such plants rapidly changes in response University, Jhongli City, Taoyuan County 320, Taiwan, ROC to biotic and abiotic stress, and this occurs not only through transcriptional control by mRNA synthesis (Agar- C.-Y. Hong Department of Agricultural Chemistry, National Taiwan wal et al. 2006; Lata and Prasad 2011; Nakaminami et al. University, Taipei 10617, Taiwan, ROC 2012; Nakashima et al. 2009; Puranik et al. 2013; Todaka

1 3 444 Plant Mol Biol (2014) 85:443–458 et al. 2012) but also through post-transcriptional control et al. 2005; Zheng et al. 2008). Knockdown of CAF1a and by mRNA degradation (Belostotsky and Sieburth 2009; CAF1b or CCR4a and CCR4b resulted in reduced cell pro- Chiba et al. 2013; Floris et al. 2009; Kojima et al. 2011; liferation (Aslam et al. 2009; Mittal et al. 2011; Morita Pruneda-Paz and Kay 2010; Rayson et al. 2012; Staiger et al. 2007). The CCR4-NOT complex also participates and Green 2011). In addition to the endonucleolytic cleav- in the mechanism involved in RNA-induced gene silenc- age process, the majority of mRNA degradation in eukary- ing. When the RNA-induced silencing complex (RISC) is otic cells is initiated by poly(A) tail shortening (Belosto- combined with 3′UTR of mRNA, CCR4-NOT complex is tsky and Sieburth 2009; Chiba and Green 2009; Meyer recruited to disrupt mRNP structure, reduce translation effi- et al. 2004). In cytosol, translatable mRNAs interact with ciency and accelerate mRNA degradation of the targeted poly(A) binding protein (PABP) and eIF4E (Caponigro and mRNA by promoting poly(A) removal in mammalian cells Parker 1995), and these mRNP complexes are disrupted (Fabian et al. 2009; Piao et al. 2010). after deadenylation; subsequently, either the 5′ cap of the All higher plants, such as Arabidopsis and rice, con- mRNA is removed and the mRNA body is degraded in a tain large numbers of CAF1 gene family members (Cai 5′–3′ direction by exoribonuclease (XRN) or the 3′ end of et al. 2011; Walley et al. 2010a, b). In the literature, only the mRNA is attacked by a 3′–5′ exosome complex (Belos- a few studies have indicated that the function of CAF1 is totsky and Sieburth 2009; Tharun and Parker 2001; Wilusz cross-related to plant tolerance to abiotic and biotic stress et al. 2001). Deadenylation in eukaryotic cell is cata- mechanisms. CaCAF1, a pepper CAF1, is required to resist lyzed by deadenylases, including PAN2-PAN3 complex, to pathogen infections (Sarowar et al. 2007). Arabidopsis DAN/PARN deadenylase (Korner et al. 1998), and CCR4- atcaf1a and atcaf1b mutants showed decreased PR1 and NOT complex (Chen et al. 2002; Tucker et al. 2001). PR2 mRNA levels, and the plants were susceptible to path- Despite the functional overlapping of the deadenylases, ogen infection (Liang et al. 2009). However, only atcaf1a the CCR4-NOT complex seems to play a major role in the mutants exhibited phenotypical salt tolerance. Additionally, mRNA deadenylation process in most eukaryotes (Parker members of the CAF1 gene, AtCAF1a to AtCAF1k, showed and Song 2004). different expression patterns in response to wounding (Wal- The CCR4-NOT complex contains two catalytic subu- ley et al. 2010a). Therefore, it is assumed that functional nits, carbon catabolite repressor 4 (CCR4) and CCR4 specificity within CAF1 families is essential for deadenyla- associated factor 1 (CAF1, also called POP2), both are tion of their mRNA targets in response to specific signals. required for deadenylation. It is believed that CCR4 is the The mRNA degradation process requires the participa- major cytoplasmic deadenylase and acts as the main cata- tion of many proteins in deadenylation, decapping, and lytic component. Evidence from yeast studies shows that exoribonucleic cleavage. These proteins are known to point mutation at catalytic residues of CCR4 abolished the colocalize with their target mRNAs in small, discrete, CCR4-NOT deadenylase function in vivo; ectopic over- and prominent granular foci known as processing bod- expression of CCR4 complements caf1 defects, whereas ies (also termed PBs or P-bodies) within the cytoplasm ectopic overexpression of CAF1 cannot complement ccr4 of eukaryotic cells (Anderson and Kedersha 2006; Eula- defects (Chen et al. 2002; Ohn et al. 2007; Tucker et al. lio et al. 2007). Deadenylation enzymes including PAN2, 2001, 2002). However, CAF1 is also essential for dead- PAN3, CCR4, and CAF1 are found to localize in P-bod- enylation in yeast, and caf1 mutant strains show defects ies (Zheng et al. 2008). Remarkably, P-bodies were unde- in deadenylation (Tucker et al. 2001, 2002). In addition, tectable in mammalian cells overexpressing a truncated studies on trypanosome and multicellular eukaryotes have CAF1 with mutations at catalytic residues; this defect can indicated that CAF1 also plays a critical role in deadenyla- be restored by coexpression of CCR4 (Zheng et al. 2008), tion, and controls the expression of certain genes involved suggesting that deadenylation is a prerequisite for P-body in vital physiological processes. Knockdown of CAF1 gene formation and mRNA decay. Arabidopsis Decapping 1 expression resulted in embryonic lethality and delayed lar- (DCP1), DCP2, VARICOSE (VCS), EXORIBIONUCLE- val developmental progress in C. elegans (Molin and Pui- ASE 4 (XRN4), PARN, and CCR4a are also colocalized sieux 2005). In Trypanosoma brucei, deletion of CAF1 in P-bodies (Moreno et al. 2013; Pomeranz et al. 2010; delayed deadenylation of bulk mRNAs, and the deadeny- Weber et al. 2008; Xu and Chua 2009), suggesting that lase activity of CAF1 was also shown to be essential for their function has been conserved from yeast to humans cell viability (Schwede et al. 2008). In Drosophila cells, the and plants through evolution. However, DCP5, identified as deletion of CAF1 but not CCR4 delayed the deadenylation an Arabidopsis P-body component and required for P-body of Hsp70 mRNA (Temme et al. 2004). Human cells contain formation and postembryonic development, is not associ- two CCR4 and two CAF1 paralogues that are all involved ated with VCS (Xu and Chua 2009). Moreover, in con- in deadenylation (Aslam et al. 2009; Funakoshi et al. trast to human and yeast eIF4E, Arabidopsis eIF4E homo- 2007; Morita et al. 2007; Schwede et al. 2008; Yamashita logue protein is localized in plant stress granules but not in

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P-bodies (Weber et al. 2008). These studies have suggested every 3 day. The hydroponically cultivated seedlings were that plant P-bodies contain different components that per- grown in a growth chamber at 28 °C with a 16-h light/8-h form divergent functions. It is still unclear whether the dark cycle. Twenty-one-day-old seedlings underwent dif- plant CAF1 is localized in P-bodies to perform deadenyla- ferent stress treatments by replacing the medium. The tion or different plant CAF1 proteins have different subcel- medium was replaced with a fresh medium prechilled to lular localizations. 4 °C (cold stress), prewarmed to 45 °C (heat stress), sup- Rice (Oryza sativa) is one of the world’s most vital crops plemented with 200 mM NaCl (salt stress) or 20 μM ABA and is the major staple food of nearly 50 % of the world’s (ABA treatment). For temperature stress treatments, the population. However, little is known about rice mRNA seedlings were transferred to growth chambers maintained deadenylation and there are no reports on the function of at 4 °C (cold stress) or 45 °C (heat stress). Drought stress rice CAF1. In this study, we cloned and characterized 4 was performed by air-drying seedlings at 28 °C, and sam- OsCAF1 genes, and showed that the recombinant OsCAF1 ples were taken after 10 and 30 % fresh weight loss was proteins exhibited deadenylase activity in vitro. The diver- recorded. For wound treatments, the leaves were punctured gent expression patterns of each member were exhibited in with needles and then cultured at 28 °C. Samples after the various tissues and were found to response to different abi- treatments were collected at indicated times, frozen in liq- otic stresses. We provide evidence to indicate that these rice uid nitrogen, and stored at 70 °C. − CAF1 proteins are localized in the cytoplasm and nucleus. We further demonstrate that OsCAF1B colocalizes with Gene expression analyses a P-body marker AtXRN4, indicating that OsCAF1B is a component of the plant P-body. Total RNA was extracted from the shoots and roots of 21-day-old seedlings treated with ABA or differential stresses by TRIzol reagent (Invitrogen, Carlsbad, CA, Materials and methods USA). RNA gel-blot analysis was performed as described (Ho et al. 2001). Ten micrograms of total RNA was sep- Identification of CAF1 homologs and phylogenetic arated on 1 % agarose gel containing 10 mM sodium analysis phosphate buffer (pH 6.5), transferred to a Hybond N1 membrane (GE Healthcare, Pittsburgh, PA, USA) and To identify the rice and maize CAF1 homologs, we used hybridized at 42 °C with a DNA probe radiolabeled by the AtCAF1a (encoded as At3g44260) sequence (Walley [α32P] dCTP. For RNA loading controls, 25S, 18S, and et al. 2010a) as a query to perform the BLAST program, 5.8S rDNA in pRY18 (Sano and Sano 1990) were excised searching the Oryza sativa and Zea mays nodes at Phyto- with BamHI and used as probes. zome (www.phytozome.net). Yeast (Saccharomyces cer- The quantitative real-time RT-PCR (qRT-PCR) analyses evisiae and Schizosaccharomyces pombe), human (Homo were performed using the FastStart Essential DNA Green sapiens), fruit fly (Drosophila melanogaster), and mouse Master (Roche, Basel, Switerland) and the iQ5 real-time (Mus musculus) CAF1 amino acid sequences were identi- PCR machine (Bio-RAD, Hercules, CA, USA). Results fied using the NCBI database (www.ncbi.nlm.nih.gov). were repeated independently at least three times and rela- Finally, all of these amino acid sequences were aligned tive gene expression was expressed as a ratio of target gene using the EBI Clustal Ω program (www.ebi.ac.uk), and the mRNA to that of rice 18S rRNA. Data were analyzed using phylogenetic trees were generated using MEGA 5 (Tamura the iQ5 2.1 software program provided by the manufac- et al. 2011). We used the neighbor-joining method to con- turer. Gene-specific primers used for qRT-PCR are listed in struct different trees with pairwise deletion. The bootstrap Table S2. calculation was performed using 1,000 replicates for statis- tical reliability. Construction of plasmids

Plant material, growth conditions, and stress treatment To construct vectors for the production of recombinant OsCAF1 proteins, full-length OsCAF1s were amplified The rice variety used in this study was Oryza sativa L. cv using PCR by gene-specific primers with cDNA derived Tainung 67 (TNG67). Seeds were surface-sterilized with from rice suspension cells. The PCR-amplified OsCAF1A, 2.5 % NaOCl for 40 min, washed extensively with sterile OsCAF1G and OsCAF1H fragments were cloned into water, and germinated in the dark on wet filter paper in pET28b (Merck KGaA, Darmstadt, Germany); OsCAF1B petri dishes at 37 °C for 48 h. After incubation, uniformly fragment was cloned into pET21a (Merck KGaA). germinated seeds were selected and cultivated in a 500 mL Expression vectors for plant cell subcellular localization beaker containing half-strength Kimura B solution replaced (Curtis and Grossniklaus 2003) were constructed using the

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Gateway cloning system as follows. The coding sequences pH 7.0, 150 mM NaCl and 10 % glycerol, and then desalted of OsCAF1, AtXRN4, AtfABP2 and AtKSS12 genes with- by HiPrep desalting FF column (GE Healthcare) and puri- out a stop codon were amplified using PCR and subcloned fied by HiPrep Sp FF column (GE heakthcare) and HiLoad into the pENTR/D-TOPO vector (Invitrogen) to generate 26/60 Superdex-75 size-exclusion column (GE Health- entry clones. Using LR clonase (Invitrogen), recombina- care). The purified proteins were enriched using centrifugal tion was conducted to transfer target gene fragments from concentrator and determined by Bradford method. entry clones to destination vectors, pMDC43, pMDC85 or pMDC139 (Brand et al. 2006; Jia et al. 2007), to generate In vitro deadenylase assay the N-terminal green fluorescent protein (GFP), C-terminal GFP or C-terminal GUS fusion constructs, respectively. The 5′-FAM-labeled RNA substrates, 5′-/56-FAM/UCU To construct the OsCAF1B-mCherry expression vec- AAAUAAAAAAA, were synthesized by Integrated DNA tor, we generated mCherry destination vector by replacing technology (Integrated DNA Technologies, Coralville, the GFP with mCherry in pMDC43 and pMDC85. Primers Iowa, USA). In vitro enzymatic assay was conducted at used for construction are listed in Table S2. 30 °C in 20 μL of a reaction buffer (50 mM Tris–Cl, pH

7.0, 50 mM NaCl, 1 mM MgCl2, 10 % glycerol) with Protein expression and purification 1 μmol RNA substrate and 1 μg of recombinant OsCAF1 proteins. After incubation, denaturing polyacrylamide gel Recombinant OsCAF1A, OsCAF1G and OsCAF1H protein electrophoresis was performed as previously described expression and purification were performed as previously (Rio et al. 2010). The RNA gel was visualized using LAS- described (Feddersen et al. 2012) with slight modifica- 4000 (GE Healthcare). tions. E. coli BL21 (DE3) containing expression plasmids were grown in 3 L LB medium with kanamycin at 37 °C Analysis of subcellular localization until a density of OD600 0.6–0.8 was reached. Recom- = binant proteins were induced with 0.5 mM IPTG and incu- The onion bulb epidermis was prepared and particle bom- bated at 20 °C overnight. Cells were harvested and resus- bardment was executed as described by (Scott et al. 1999) pended using Buffer A (50 mM Tris–HCl, pH 7.0, 300 mM to introduce GFP, mCherry or GUS expression vectors

KCl, 5 mM MgCl2, 5 mM β-mercaptoethanol (BME), using a PDS-1000 biolistic device (Bio-Rad) at 1,100 p.s.i. 20 mM imidazole, 10 % glycerol and 1 mM PMSF) and Bombarded specimens were incubated on MS solid lysed by sonication. The crude extract was centrifuged at medium for 6 h, and were then observed using an Olym- 12,000 rpm at 4 °C, and then passed through a 0.45-μm pus IX71 (Olympus, Tokyo, Japan) inverted fluorescence nonpyrogenic filter. The protein solution was loaded on a microscope. Confocal observation was conducted using a HisTrap column (GE Healthcare) preequilibrated in Buffer Zeiss LSM710 microscope (Carl Zeiss Microscopy GmbH, A, and then washed with Buffer B (50 mM Tris–HCl, pH Jena, Germany). Serial optical sections of images were

7.0, 1 M KCl, 5 mM MgCl2, and 5 mM BME), and eluted captured on a Zeiss LSM710 microscope. with Buffer C (containing 500 mM imidazole in Buffer A). For analysis of GUS activity, bombarded onion bulb epi- The elution was dialyzed using buffer D (50 mM Tris–HCl, dermis were incubated on MS solid medium for 6 h and pH 7.0, 200 mM NaCl, 1 mM MgCl2, 1 mM BME, 10 % were then incubated in GUS staining solution at 37 °C glycerol). The recombinant proteins were further purified overnight, as described by Jefferson et al. (1987). The by gel filtration using HiLoad 26/60 Superdex-75 size- specimens were observed and photographed using a Zeiss exclusion column (GE Healthcare) with 50 mM Tris–HCl LSM510 microscope (Carl Zeiss Microscopy GmbH). (pH 7.0), 50 mM NaCl, 1 mM BME, 10 % glycerol. Frac- Images were analyzed using Zeiss LSM Image Browser tion containing rOsCAF1A, rOsCAF1G or rOsCAF1H was software. concentrated using a Vivaspin centrifugal concentrator (GE Healthcare), and soluble protein was determined using the Bradford method (Bradford 1976). Results For OsCAF1B protein expression, E. coli. Rosetta2 (DE3) containing OsCAF1 B expression plasmid was Four expressed CAF1 genes were identified from rice grown in 3 L LB medium with kanamycin at 25 °C until OD600 0.6–0.8 was reached. Recombinant proteins To identify rice CAF1 homologs, we used the protein = were induced with 0.5 mM IPTG and incubated at 20 °C sequence AtCAF1a as a query for the basic local alignment overnight. After sonication, centrifugation and filtration, search at Phytozome (www.phytozome.net). Eighteen putative the total proteins were precipitated by ammonium sulfate homologs (OsCAF1A-R) were identified from rice genome (20–40 %). The pellet was dissolved in 50 mM Tris–HCl, database (Table S1). However, only four corresponding

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Fig. 1 Amino acid sequence alignments of CAF1 proteins. CAF1 red. A fifth residue, histidine, located before the final conserved D, is protein sequences of rice (OsCAF1), maize (ZmCAF1), Arabidop- also highly conserved and labeled in blue. The budding yeast POP2 sis (AtCAF1), human (HsCNOT7/8), fruit fly (DmCAF1), budding diverges in the first and final residues indicated in green, and the con- yeast (ScPOP2), and fission yeast (SpCAF1) were aligned using sensus DEDD is changed to SEDQ. The number of amino acid resi- ClustalΩ and edited using GeneDOC. Three conserved DEDD motifs dues from N-, C-termini or between conserved blocks are indicated in are indicated at the top. Conserved residues, DEDD, are labeled in parentheses

cDNA or EST (expressed-sequence tag) sequences of these human, mouse, fruit fly, and budding and fission yeast putative OsCAF1s could be obtained from NCBI and Plant- CAF1 families. The phylogenetic tree (Fig. 2) showed GDB (www.plantgdb.org) database. These four homologs, that all of the Arabidopsis CAF1 proteins could be divided encoded by LOC_Os08g34170, LOC_Os04g58810, into three groups, consistent with a previous report (Wal- LOC_Os09g24990 and LOC_Os02g55300, were named ley et al. 2010a). The four rice CAF1 proteins analyzed OsCAF1A, OsCAF1B, OsCAF1G and OsCAF1H, respec- in this study were clustered into two groups, I and III. tively (Table S1). Using the same way to search, we identified OsCAF1A, OsCAF1G, OsCAF1H were clustered in Group 21 AtCAF1a related sequences in maize (Zea mays). Among III, whereas OsCAF1B and its counterparts, AtCAF1a and them, ZmCAF1A.1 (GRMZM2G047019), ZmCAF1A.2 AtCAF1b, were clustered in the same clade, Group I. The (GRMZM2G123328), ZmCAF1G (GRMZM2G071059), amino acid sequences of the OsCAF1 proteins in Group III ZmCAF1H.1 (GRMZM2G179633), and ZmCAF1H.2 were more similar to ZmCAF1 than to AtCAF1 proteins. (GRMZM2G110960) cDNA sequences were available in public databases. The amino acid sequence comparison anal- Recombinant OsCAF1 proteins contain deadenylase ysis indicated that the rice and maize CAF1 homologues we activity in vitro identified are well conserved at RNase D domain with three conserved motifs and four important catalytic residues of To determine whether the OsCAF1 proteins contained nuclease activity, DEDD (Fig. 1). In addition, these CAF1 deadenylation activity, the recombinant His–OsCAF1 homologues contain the fifth conserved amino acid residue, proteins were expressed in E. coli. Three recombinant histidine (Fig. 1). Therefore, all of the identified sequences His–OsCAF1 proteins, rOsCAF1A, rOsCAF1G, and from rice and maize were grouped in the CAF1 family (ID: rOsCAF1H, were obtained by HisTrap and gel filtra- PF04857.15). tion column. Due to a protein aggregation problem of A phylogenetic relationship was constructed using rOsCAF1B protein, we performed ammonium sulfate pre- amino acid sequences from rice, maize, Arabidopsis, cipitation, followed by cation-exchange and gel filtration

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Fig. 2 Phylogenetic relation- ships of CAF1 family members. Phylogenetic tree among rice, maize, Arabidopsis, human, mouse, fruit fly, budding and fission yeast CAF1 is generated III by MEGA 5 and rooted with SpCAF1 and bootstrap values (>50) are indicated at each node. The members of CAF1 from rice, maize, and Arabidop- sis are categorized into Groups, I, II, and III, with at least 50 % bootstrap support. Accession numbers of genes listed here are depicted in the Supplementary Table S2

II

I

to purify intact rOsCAF1B. The deadenylase activities of completely digest the substrates (Fig. 3a, d and e). To deter- purified proteins were analyzed by incubating them with mine whether the rOsCAF1B exhibited stepwise dead- 5′-FAM-labeled RNA substrates for various lengths of enylase activity as other rOsCAF1 proteins, less amount time. Reaction mixtures were subjected to polyacrylamide of rOsCAF1B was examined. As result shown in Fig. 3c, gel electrophoresis; the 5′-FAM-labeled fragments were tenfold diluted OsCAF1B proteins (0.1 μg) removed the then visualized using fluorometer. rOsCAF1A, rOsCAF1G first seven adenine nucleotides of the substrates by step- and rOsCAF1H proteins were able to remove the first wise shortening. These results indicated that the direction seven adenine nucleotides of the substrates within 1 min of RNA degradation was from 3′ to 5′. The intermediate by stepwise shortening (Figs. 3a, d and e). Compared with products (UCUAAAU) accumulated in all of the reaction rOsCAF1A, rOsCAF1G and rOsCAF1H, the substrates mixtures. The OsCAF1 proteins removed the uridine nucle- were digested completely by rOsCAF1B within 0.5 min otide and the following three adenine nucleotides and pro- (Fig. 3b), whereas others required longer than 3 min to duced shorter products FAM/UCU (Fig. 3a–e). To confirm

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(A) (D) rOsCAF1A rOsCAF1A(m) rOsCAF1G rOsCAF1G(m) Time Time RNaseA 0 0.5 1320 10 20 RNaseA 020.5 1320 10 0 (min) (min)

UCUAAAU UCUAAAU UCU UCU

(B) (E) rOsCAF1B rOsCAF1B(m) rOsCAF1H rOsCAF1H(m) Time Time

0 0.5 1320 10 20 RNaseA 020.5 1320 10 0 RNaseA (min) (min)

UCUAAAU UCUAAAU UCU UCU

(C) rOsCAF1B (10X) Time

0 0.25 0.5 13 5 10 RNaseA (min)

UCUAAAU UCU Probe: FAM-UCUAAAUAAAAAAA

Fig. 3 Deadenylase activity assay of recombinant OsCAF1 pro- (f) were used in parallel time-course reactions. The reaction prod- teins. Purified rOsCAF1A (a), rOsCAF1B (b), tenfold diluted ucts were subjected to polyacrylamide gel electrophoresis. The FAM rOsCAF1B (c), rOsCAF1G (d), rOsCAF1H (e) and a catalytic site images were obtained using a fluorescence imaging system. Commer- mutants, rOsCAF1A(m) (a), rOsCAF1B(m) (b), rOsCAF1G(m) (d) cial RNase A was used as positive control and rOsCAF1H(m) (e) with 5′-FAM labeled synthetic RNA probe

Seedlings Mature plants that the deadenylase activity came from the OsCAF1 pro- teins, a mutation strategy at the conserved active site resi- dues was applied. Four mutated proteins consequently did not exhibit deadenylase activity (Fig. 3a–e), indicating OsCAF1A that the deadenylase activity was not from contaminating OsCAF1B nucleases during purification. These results indicated that OsCAF1 proteins are active 3′–5′exonucleases. OsCAF1G

rRNA Unique and overlapping expression patterns among members of rice CAF1 family Fig. 4 Expression patterns of OsCAF1 genes in rice. Total RNAs were isolated from roots and shoots of 21-day-old seedlings, and To determine the expression profile of the OsCAF1 family from various tissues of 3-month-old plants. Northern blot analysis in rice, total RNAs were isolated from various tissues and was performed using the OsCAF1A-, OsCAF1B-, OsCAF1G- and subjected to Northern blot analysis. In the shoots of 21-day- OsCAF1-specific cDNA regions and 28S rRNA gene as probes. Two bands detected using the OsCAF1B probe are indicated by arrows old seedlings and the sheaths of 3-month-old mature plants, as OsCAF1B (L) and OsCAF1B (S), respectively. The mRNA of OsCAF1A and OsCAF1G mRNA were slightly detectable, OsCAF1H was not detectable and data are not shown

1 3 450 Plant Mol Biol (2014) 85:443–458 and were significantly expressed in the green leaves, senes- Differential subcellular localization of OsCAF1 proteins cent leaves, panicle axes, and spikelets of mature plants (Fig. 4a). Two different transcripts of OsCAF1B mRNA, The cellular localization of POP2 (yeast CAF1) and OsCAF1B (L) and OsCAF1B (S), were detected as inverse CNOT8 (human CAF1) occurs predominantly in the cyto- expression patterns in the various tissues. The OsCAF1B plasm and P-body (Teixeira and Parker 2007; Tucker et al. (L) mRNA was detected in roots, sheaths, nodes, and spike- 2001; Yamashita et al. 2005; Zheng et al. 2008). Another lets, whereas OsCAF1B (S) was detected in leaves and human CAF1 homolog, CNOT7, is located in the cyto- senescent leaves (Fig. 4). Northern blot analysis detected plasm and nucleus (Robin-Lespinasse et al. 2007). Amino no OsCAF1H mRNA in any of the selected tissue (data not acid sequence analysis indicated that some OsCAF1 pro- shown). teins have various numbers of putative NLS and NES To examine whether the expression of each OsCAF1 (Fig. 6a). To determine the subcellular localizations of gene responses to various abiotic stresses, Northern blot OsCAF1 proteins, OsCAF1 genes were fused with GFP analysis and qRT-PCR were used to determine the mRNA under the control of a 35S promoter and introduced into levels of OsCAF1 in the roots and shoots of 2-week-old onion epidermal cells using particle bombardment. To pre- rice seedlings subjected to abscisic acid (ABA) (20 μM), vent artifacts resulting from high protein expression, GFP- drought (air drying), cold (4 °C), salt (200 mM NaCl), and emitted fluorescence signals were observed at 6 h after heat (45 °C) treatments. The accumulation of OsCAF1A bombardment. As shown in Fig. 6b, fluorescence signals mRNA was slightly induced by ABA, salt, cold, and heat from GFP fused to the C terminal of each OsCAF1 pro- both in roots and shoots (Fig. 5a). The level of OsCAF1B tein (OsCAF1–GFP) were present not only in the nucleus (L) mRNA in roots and OsCAF1B (S) mRNA in shoots was but also in the cytoplasm, the same cellular localiza- significantly induced by drought stress (with 10 or 30 % tion pattern as GFP only. In the cytoplasm, OsCAF1A–, water loss) and cold stress (Fig. 5a). We further quanti- OsCAF1G– and OsCAF1H–GFP protein signals were fied the relative levels of OsCAF1B mRNA, including both distributed uniformly (Fig. 6b), whereas OsCAF1B (L)– forms, in plants subjected to drought and cold stresses. GFP was localized to specific cytosolic foci and formed Consistent with the Northern blot analysis, OsCAF1B filamentous structures (Figs. 6b, c). Similar subcellular mRNA levels increased approximately more than 8.5-fold localization patterns were observed when GFP fused to the in drought-stressed shoots, when the plants lost more than N-terminus of each OsCAF1 (GFP–OsCAF1) (Supplemen- 10 % of their fresh weight in water (Fig. 5b). The level of tal Figure 1).We further examined the subcellular locali- OsCAF1B mRNA increased 2.2- and 9.4-fold after shifting zation of OsCAF1 proteins by fusing another reporter, to 4 °C for 8 h in roots and shoots, respectively (Fig. 5c). It β-glucuronidase (GUS), in onion epidermal cells. Each has been reported that Arabidopsis AtCAF1a and AtCAF1b OsCAF1 fusion proteins (OsCAF1–GUS) were distributed mRNA accumulated rapidly within 15 min after ABA and both in the nucleus and cytoplasm, whereas GUS was pre- wounding treatments (Liang et al. 2009). Since OsCAF1B is dominantly localized in the cytoplasm (Supplemental Fig- closely related to AtCAF1a and AtCAF1b from phylogenic ure 2). The OsCAF1B–GUS was also localized to specific tree, we determined whether the expression of OsCAF1B cytosolic foci and filamentous structures. In contrast to the was in response to ABA and wounding treatments. In seed- well-distributed signals of GFP and OsCAF1A–GFP in the lings, the accumulation of OsCAF1B mRNA drastically nucleus, the OsCAF1B (L)–, OsCAF1G– and OsCAF1H– increased within 15 min after wounding and ABA treat- GFP signals in the nucleus exhibited dense spots that were ments, but then declined 1 h after treatments (Fig. 5d, e). localized to the nucleoli (Fig. 7). These results indicated that OsCAF1B transcripts were transiently induced by drought, cold, ABA, and wounding OsCAF1B (L) is colocalized with P‑body marker treatments. OsCAF1G mRNA was detected in shoots and and tubulin marker was slightly repressed by heat stress, ABA and cold stress (Fig. 5a). Moreover, OsCAF1G mRNA was detectable Specific localization in the cytosolic foci and filamentous under ABA, drought, salt and heat stress in roots. Among structures of OsCAF1B was observed (Fig. 6b, c). Since the different stress treatments, OsCAF1H mRNA was only CNOT7, a human CAF1 homolog, is known to localize detected under heat stress, and levels of OsCAF1H mRNA in P-bodies (Zheng et al. 2008), we determined whether increased 9.9-fold in roots and 4.2-fold in shoots after shift- OsCAF1B (L) was associated with P-bodies, and con- ing to 45 °C for 1 h, and dramatically decreased to the basal ducted colocalization experiments using the Arabidopsis level after 3 h of recovery at room temperature (Fig. 5f). P-body marker EXONUCLEASE 4 (AtXRN4) (Moreno The differential expression patterns of rice CAF1 family et al. 2013; Souret et al. 2004; Weber et al. 2008). Con- members indicate that each OsCAF1 gene in rice may have sistent with Arabidopsis protoplasts, AtXRN4–GFP a unique function in response to various abiotic stresses. was localized in cytoplasmic foci resembling P-bodies

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(A) Roots Shoots

OsCAF1A

OsCAF1B (L) OsCAF1B OsCAF1B (S)

OsCAF1G

OsCAF1H

rRNA

(B) (C) (D)

OsCAF1B OsCAF1B OsCAF1B Relative mRNA level Relative mRNA level Relative mRNA level

C 10% 30% 0816 24 36 48 00.251 6 FW lose Cold, 4 (hours) Wounding (hours)

(E) (F) OsCAF1H OsCAF1B Relative mRNA level Relative mRNA level

00.251 612 01313 20 µM ABA (hours) Heat, 45 Recovery, 25 (hours) (hours)

Fig. 5 Expression patterns of OsCAF1 genes under various stress with ethidium bromide staining. b–f Total RNAs were isolated from treatments. a Two-week-old rice seedlings subjected to ABA 2-week-old rice seedlings treated by applying drought, cold, ABA, (20 μM), drought (air dry), cold (4 °C), salt (200 mM NaCl), and wounding and heat in short periods and subjected to qRT-PCR analy- heat (45 °C) were treated for various periods. Northern blot analysis sis by using primers specific for OsCAF1B (b–e) and OsCAF1H (f). of total RNAs isolated from rice plants were detected using probes Relative mRNA expression levels were normalized to the rice 18S from the OsCAF1A-, OsCAF1B-, OsCAF1G- and OsCAF1H-spe- rRNA gene cific cDNA regions. Ribosomal 25S and 18S RNAs were detected

1 3 452 Plant Mol Biol (2014) 85:443–458

(A) (B) Fluorescence Merge

GFP

OsCAF1A– GFP

(C) OsCAF1B (L)–GFP OsCAF1B (L)– GFP

OsCAF1G– GFP

OsCAF1H– GFP

Fig. 6 Subcellular localization of OsCAF1–GFP fusion proteins tain a full-length coding region without a stop codon of the OsCAF1 in onion epidermal cells. a Schematic diagram of rice CAF1 pro- genes fused to GFP, by particle bombardment. The GFP along is used teins. The gray boxes indicate the CAF1 conserved regions. Puta- as the control. c The granules and filamentous structures assembled tive nuclear localization signals (NLS) and nuclear exclusion signals by OsCAF1B (L)–GFP were detected in the cytoplasm. The green (NES) of OsCAF1 proteins are indicated as white and black blocks. fluorescent signals were obtained using a florescence microscope and b The rice OsCAF1A–, OsCAF1B (L)–, OsCAF1G–, and OsCAF1– are shown in green, and nuclei are indicated by white arrows. Scale GFP fusion proteins are localized to the nucleus and cytoplasm. bar 50 μm Onion epidermal cells were transformed using constructs, which con-

(Fig. 8a). OsCAF1B (L)–mCherry fluorescent fusion Discussion proteins were colocalized with AtXRN4–GFP, indicating that OsCAF1B (L) localized in P-bodies (Fig. 8a). How- The CCR4-NOT complex is known to function in dead- ever, certain OsCAF1B (L)–mCherry proteins were not enylation, and has been proposed to share a highly con- colocalized with AtXRN4–GFP. By contrast, OsCAF1B served mechanism of enzymatic action among eukaryotes. (L)–GFP proteins assembled as filamentous structures However, the roles of the individual components of the in the cytoplasm are probably microfilaments or micro- CCR4-NOT complex are not completely consistent among tubules (Fig. 6b, c). We examined whether OsCAF1B different species. The deadenylation activity of CAF1 (L)–mCherry was associated with a microtubule marker, is not essential in yeast, whereas it plays a pivotal role in AtKSS12, Arabidopsis katanin domain one and two yeast and animal cells (Schwede et al. 2008; Temme et al. (Stoppin-Mellet et al. 2007), or a microfilament marker, 2004; Tucker et al. 2002). In Entamoeba histolytica, the AtfABP2, the second actin-binding domain of Arabidop- CCR4 homolog gene is not even detectable (Lopez-Rosas sis fimbrin. The results shown in Fig. 8b indicated that et al. 2012). Higher plants contain a number of CAF1 gene OsCAF1B (L)–mCherry and AtKSS12-GFP were colo- family members, unlike animals and yeasts which contain calized, wherease AtfABP2-GFP were not (Supplemen- only one or two CAF1 genes. In this study, we identified tal Figure 3), indicating that OsCAF1B (L) is associated four homologous rice CAF1 genes and demonstrated that with microtubules. recombinant rice CAF1 proteins exhibited deadenylase

1 3 Plant Mol Biol (2014) 85:443–458 453

Fig. 7 The OsCAF1B (L)–, Fluorescence Bright Merge OsCAF1G– and OsCAF1H– GFP fusion proteins are local- ized to the nucleolus. Nuclei images of the onion epider- mal cells transformed using GFP OsCAF1A-, OsCAF1B (L)– OsCAF1G– and OsCAF1H– GFP constructs. The GFP signals were aggregated to form small granules in nucleoli, observed using a florescence microscope. White arrows indi- cate nucleoli. Scale bar 50 μm OsCAF1A– GFP

OsCAF1B (L)– GFP

OsCAF1G– GFP

OsCAF1H– GFP

activity. We also analyzed rice CAF1 gene expression OsCAF1G, and OsCAF1H are expressed in rice. However, and subcellular localization patterns to offer new insights we cannot rule out the possibility that other OsCAF1 genes for exploring the functional divergence of the OsCAF1 could be detected under particular conditions. gene family during rice growth and development, and in Amino acid sequence alignment and phylogenetic analy- response to various stresses. sis clearly divided these four expressed OsCAF1 proteins Bioinformatics analysis revealed 8–16 CAF1 homolo- into two groups. Regarding classification, OsCAF1A, gous genes in the rice genome (Walley et al. 2010b). In this OsCAF1G, and OsCAF1H are Group III CAF1 proteins. study, we found 18 putative CAF1 genes in rice genome Orthologous pairs of rice and maize CAF1 members are database. However, only OsCAF1A, OsCAF1B, OsCAF1G more prevalent than others in the phylogenetic tree, sug- and OsCAF1H transcripts were identified from rice cDNA gesting that ancestor CAF1 genes were shared before the or EST sequence database. In addition, no OsCAF1C to divergence of maize and rice. Within Group III, the subcel- OsCAF1F transcripts in rice suspension cultured cells lular localization patterns of OsCAF1A, OsCAF1G and were able to detect by RT-PCR analysis (data not shown). OsCAF1H were also similar. However, the expression pat- Therefore, it is probable that only OsCAF1A, OsCAF1B, terns of individual OsCAF1 genes differed in the course of

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(A) OsCAF1B-mCherry AtXRN4-GFP Bright Merge

mCherry AtXRN4-GFP Bright Merge

(B)

OsCAF1B -mCherry

AtKSS12 -GFP

Merge

Fig. 8 The OsCAF1B (L) is colocalized to P-bodies and tubulin mCherry-only construct was used as a control and was not colocal- fibers. a Onion epidermal cells were transformed using OsCAF1B ized with AtXRN4. (b) Onion epidermal cells were transformed with (L)–mCherry and AtXRN4–GFP constructs. The mCherry and OsCAF1B (L)–mCherry and AtKSS12–GFP. The mCherry and GFP GFP images were observed using a florescence microscope. Colo- images were obtained using a confocal microscope. Fibers containing calization of OsCAF1B (L)–mCherry and AtXRN4–GFP were OsCAF1B (L)–mCherry (red image) were colocalized with a tubulin detected in merged images (yellow spots). Some granules indicated marker, AtKSS12–GFP (green image). Scale bar 50 μm by white arrows were not located with AtXRN4 (red spots). The

1 3 Plant Mol Biol (2014) 85:443–458 455 development and in response to various abiotic stresses. 2008). Although it is still unclear whether the localization For example, OsCAF1A and OsCAF1G were predomi- of CAF1 in P-bodies is important for mRNA deadenyla- nately expressed in mature plant green tissues; OsCAF1G tion, this study showed that one rice CAF1 member was mRNA was not detectable in the roots of seedlings; localized in P-bodies and may play a certain role there. OsCAF1H was induced only by heat stress. Thus, although We found that more cytosolic foci were formed by functional redundancies among OsCAF1s are extensive, OsCAF1B (L)–mCherry than AtXRN4-GFP, indicating individual OsCAF1 genes may play specific roles dur- that OsCAF1B could be a component of granules other ing biological processes such as sub- or neofunctionaliza- than P-bodies. Arabidopsis CCR4a is known to locate tion of individual CAF1 protein in plants as described by both in P-bodies and siRNA-bodies (Moreno et al. 2013). Walley et al. (2010a, b). By contrast, OsCAF1B was clus- Additionally, XRN1, eIF4E, tristetraprolin (TTP), and oth- tered in Group I with the biotic and abiotic stress-related ers have been shown to be components of P-bodies and proteins, AtCAF1a and AtCAF1b. Moreover, the transcript stress granules (Kedersha et al. 2005). In addition to the levels of OsCAF1B peaked at 15 min and decreased rap- granule structure, filamentous structures in the cytoplasm idly in response to wounding, consistent with AtCAF1a and are assembled by OsCAF1B (L)–GFP and OsCAF1B AtCAF1b (Liang et al. 2009). This suggests that the mecha- (L)–mCherry. The OsCAF1B (L) filamentous structures nism regulating the expression of the response to wounds were colocalized with microtubules. Recently, P-bodies exhibited by OsCAF1B, AtCAF1a, and AtCAF1b was con- and stress granules have been reported for their asso- served and derived from the same ancestor. ciation with microtubules (Aizer et al. 2008; Loschi et al. The two transcript forms of OsCAF1B, OsCAF1B (L) 2009; Sweet et al. 2007). Further study will be required to and OsCAF1B (S), were observed in Northern blot analy- address whether OsCAF1B (L) locates to stress granules sis, and their tissue expression patterns did not overlap but and siRNA-bodies; such study will provide insights into the were both induced by cold, drought, and ABA. Further- dynamic function of rice CAF1 members. more, genomic Southern blot analysis using the OsCAF1B The CCR4-NOT complex has been shown to participate coding region as a probe was able to detect only one in many processes in the nucleus, including transcription, hybridization signal (Supplementary Figure 4). Therefore, DNA repair, histone modification, chromatin remodeling, we assume that these two OsCAF1B transcripts are derived nuclear RNA quality control, and mRNA export (Collart from the same gene LOC_Os04g58810. Two forms of and Panasenko 2012; Jayne et al. 2006; Mulder et al. 2005, OsCAF1B cDNA clones, 006-203-G11 (OsCAF1B (L)) and 2007; Zwartjes et al. 2004). However, most components of J023025J17 (OsCAF1B (L)) are reported in public avail- CCR4-NOT are detected predominantly in the cytoplasm able database. The deduced amino acid sequence of puta- in yeasts, mammals, fruit flies, and trypanosomes, in addi- tive OsCAF1B (S) cDNA contains the conserved RNase D tion to the nucleus (Lau et al. 2010; Schwede et al. 2008, motif, but not the second NLS (Supplementary Figure 5). 2009; Temme et al. 2010; Tucker et al. 2001; Yamashita However, we were not able to clone OsCAF1B (S) cDNA et al. 2005). OsCAF1–GFP fusion proteins may dif- successfully from neither leaves, calli nor suspension cul- fuse passively into the nucleus, but functions of OsCAF1 tured cells of rice. Future studies to determine whether proteins in the nucleus are worthy to study. In addition, OsCAF1B encodes two isoforms of OsCAF1B proteins OsCAF1B (L), OsCAF1G, and OsCAF1H (except to might provide more information on the specificity of the OsCAF1A) were detected in the nucleus with dense spots OsCAF1B function in rice. localized in the nucleolus, implying that rice CAF1 pro- In our subcellular localization analysis, fluorescent teins may participate known biogenesis and processing of reporter proteins fused to OsCAF1A, OsCAF1G, or rRNA in the nucleolus. Alternatively, CAF1 might partici- OsCAF1H did not form any granule-like structures within pate in RNA quality control, a process closely related to the cytoplasm. The uniform distribution of these three the nucleolus and poly(A) tails, including snRNA, tRNA, OsCAF1 proteins in the cytoplasm is consistent with yeast rRNA, and pre-mRNA. A previous study reported that CAF1 proteins that do not form punctate structures under a physical interaction and functional connection exists normal conditions. However, yeast CAF1 can be recruited between CCR4-NOT and the TRAMP complex (Azzouz to P-bodies under glucose-deprived or stress condition et al. 2009), which is involved in the quality control of (Teixeira and Parker 2007). Considering the differential snoRNAs, snRNA, tRNA, rRNA and pre-mRNA (Azzouz expression patterns of each rice CAF1 member, we pro- et al. 2009; Dez et al. 2006; Doma and Parker 2007; Ege- pose that OsCAF1A, OsCAF1G, and OsCAF1H may re- cioglu et al. 2006; Fang et al. 2004; Kadaba et al. 2004; localize to P-bodies under specific conditions; for example, LaCava et al. 2005; Wyers et al. 2005). Mutations in CAF1 OsCAF1H under heat stress. However, OsCAF1B (L) was and other components of the CCR4-NOT complex could clearly localized in P-bodies, similar to CAF1 in human potentially cause the accumulation of polyadenylated and Drosophila cells (Temme et al. 2010; Zheng et al. snoRNAs (Azzouz et al. 2009). Future studies will be

1 3 456 Plant Mol Biol (2014) 85:443–458 necessary to determine whether this model can support Chiba Y, Green PJ (2009) mRNA degradation machinery in plants. J rice CAF1 action in the nucleolus. Plant Biol 52(2):114–124 Chiba Y, Mineta K, Hirai MY, Suzuki Y, Kanaya S, Takahashi H, CCR4-NOT complex contains several subunits that have Onouchi H, Yamaguchi J, Naito S (2013) Changes in mRNA sta- been demonstrated to play critical roles for CCR4-NOT bility associated with cold stress in Arabidopsis cells. Plant Cell complex in yeast and mammalian cells. Currently, the role of Physiol 54(2):180–194 CCR4, a deadenylase component of CCR4-NOT complex, Clark LB, Viswanathan P, Quigley G, Chiang YC, McMahon JS, Yao G, Chen J, Nelsbach A, Denis CL (2004) Systematic is not well investigated in plants. There are several putative mutagenesis of the leucine-rich repeat (LRR) domain of CCR4 genes of CCR4 homologs in rice and Arabidopsis (Dupres- reveals specific sites for binding to CAF1 and a separate criti- soir et al. 2001; Winkler and Balacco 2013). However, their cal role for the LRR in CCR4 deadenylase activity. J Biol Chem deduced amino acid sequences are less conserved in N-ter- 279(14):13616–13623 Collart MA, Panasenko OO (2012) The Ccr4–not complex. Gene minal region of CCR4 in yeast and mammalian cells. For 492(1):42–53 example, plant CCR4 homologs lack the leucine-rich repeat Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for motif that is required for interaction with CAF1 proteins in high-throughput functional analysis of genes in planta. Plant yeast and mammalian cells (Clark et al. 2004; Mittal et al. Physiol 133(2):462–469 Dez C, Houseley J, Tollervey D (2006) Surveillance of nuclear- 2011). 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