A nucleosome assembly protein-like polypeptide binds to chloroplast gr
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《核酸研究医学期刊》
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universit?t Bochum D-44780 Bochum, Germany
*To whom correspondence should be addressed. Tel: +49 234 3226212; Fax: +49 234 3214184; Email: ulrich.kueck@ruhr-uni-bochum.de
ABSTRACT
In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.
INTRODUCTION
Group II introns have been detected in eukaryotic organelles as well as in eubacterial genomes as intervening sequences of protein, tRNA or rRNA coding genes. Common to all group II introns is a conserved secondary structure that consists of six double-helical domains (DI-DVI) radiating from a central wheel (1). Group II introns splice via two sequential transesterifications that in vitro occur in some cases autocatalytically. In vivo, however, splicing is dependent on protein co-factors as was shown by mutant analyses (2,3).
In Chlamydomonas reinhardtii chloroplasts, two trans-splicing introns were found in the psaA gene encoding the major P700 chlorophyll a/b-binding protein (4). In the case of the first intron of the psaA gene, three independently transcribed RNAs associate via tertiary interactions to form a functional group II intron, resulting in trans-splicing of the flanking exon 1 and exon 2 (5). Part of this tripartite group II intron is the tscA RNA that is processed from a chloroplast-encoded precursor RNA. Secondary structure predictions revealed that tscA contains domain DII and DIII as well as partial domains DI and DIV of the conserved secondary group II intron structure (6).
Mutant work in C.reinhardtii has shown that >14 nuclear genes affect the chloroplast trans-splicing reaction, and complementation of some of the characterized mutants led to the identification of the molecular determinants. Although some of the identified polypeptides are related to proteins involved in nucleic acid metabolisms, this function seems to be non-essential for intron splicing (7,8). Similar to nuclear mRNA splicing, these protein components might be part of a chloroplast splicing complex, because they were found in large stromal or membrane-bound RNA-containing complexes (9–11).
To define further components of a putative ‘chloroplast spliceosome’ in C.reinhardtii, we used the tscA RNA as bait to identify novel intron RNA-binding proteins. Using the yeast three-hybrid system, we isolated a polypeptide that seems to be a novel member of the multifunctional nucleosome assembly protein (NAP) family. These well-conserved eukaryotic histone chaperones facilitate, for example, the nucleosome assembly and remodelling of chromatin, and are implicated in transcriptional regulation and cell cycle regulation (12,13). Here, we assign a novel function to a NAP-like protein. Laser scanning confocal fluorescence microscopy (LSCFM) and in vitro binding assays demonstrate that chloroplasts of C.reinhardtii contain a NAP-like protein that specifically binds to organellar group II intron RNA and U-rich chloroplast transcripts. Our data further support the view that during evolution, chloroplasts have acquired novel nuclear components that most probably were not delivered by endosymbiont gene transfer (14).
MATERIALS AND METHODS
Strains, culture conditions and transformation
C.reinhardtii cell wall depleted, arginine auxotroph strain argb-ecw15 as well as wild-type strain CC406 cw15 mt- were obtained from the Chlamydomonas Center (Duke University, Durham, NC, USA) and were grown as described on Tris–acetate–phosphate (TAP) medium (15). When required, the medium was supplemented with 50 μg arginine per ml. Nuclear transformation was carried out using the glass bead method (16) and was performed according to (8) with the following modifications. A total of 1–3 x 106 cells/ml were incubated with 1–5 μg supercoiled or SpeI digested pMF59 (17) plasmid DNA. After transformation, cells were spread on solid TAP medium and incubated in continuous light at 25°C. For selection of transformants carrying pMF59, medium contained 5 μg/ml of zeocin (Invitrogen, Karlsruhe, Germany).
Recombinant plasmids
All recombinant plasmids used for in vitro transcription, PCR analysis, protein synthesis, yeast three-hybrid screening, hybridization or generation of transgenic algal strains are listed in Table 1. Sequences of all oligonucleotides are given in Table 2.
Table 1 Plasmids used in this work
Table 2 Oligonucleotide primer pairs used in PCR and RT–PCR experiments
Molecular biological techniques
Procedures for standard molecular techniques were performed as described previously (18). Escherichia coli strain XL1-blue MRF' served as host for general plasmid construction and maintenance (19). The C.reinhardtii cDNA library contains double-stranded cDNA fragments with EcoRI adaptors cloned into the EcoRI site of pGAD10 in DH10B (BD Biosciences Clontech, Heidelberg, Germany) (20). pGAD10 derivatives were used in the yeast three-hybrid screen. C.reinhardtii total RNA was prepared according to (4) and RNA blot experiments were carried out according to (8). Northern analyses were performed with a radioactively labelled 1690 bp pGAD6-19/EcoRI cNapl-specific probe and a 460 bp pGAD-Rps18/EcoRI–BamHI Rps18-specific probe. cDNA was prepared using OmniscriptTM Reverse Trancriptase (Qiagen, Hilden, Germany) and purified with NucAwayTM Spin Columns (Ambion, Austin, TX, USA) according to the manufacturer's protocols. For PCRs, Taq polymerase (Eppendorf, Hamburg, Germany), Hot MasterTM Taq DNA Polymerase (Eppendorf) and the Expand Long Template PCR System (Roche, Mannheim, Germany) were used. Oligonucleotide primers applied for conventional PCR are listed in Table 2.
The yeast three-hybrid system
The yeast three-hybrid analysis was performed as described previously (8). Plasmids are listed in Table 1. Plasmids pADRevM10 and pRevR2 were generous gifts from Dr U. Putz (University of Hamburg) (21). Plasmid pRevtscA1 (8) was used to screen a C.reinhardtii cDNA library (Table 1).
Electrophoretic mobility shift assays (EMSAs)
Recombinant plasmid pQE70-cNAPL (see above) was used for synthesis of cNAPL in E.coli. Recombinant His-tagged cNAPL protein was purified from E.coli with Ni2+-NTA-agarose resin according to the manufacturer's protocols (Qiagen). For RNA mobility shift assays, uniformly 32P-UTP-labelled run-off transcripts served as substrate RNAs and were generated by in vitro transcription of plasmids as given in Table 1. In vitro transcription and EMSAs were performed as previously reported (22,23). Unlabelled competitor RNAs and non-specific competitor RNA, derived from plasmid pBIIKS+, were synthesized as described (22). DNA mobility shift assays were done essentially as described previously (24) with the following modifications. The probes were amplified by PCR with appropriate plasmid DNA or genomic DNA of strain arg-cw15 in the presence of dATP. The labelled probes were purified by denaturing PAGE. Binding reactions were carried out by incubating 30 fmol of the probes with 15 μg of recombinant cNAPL. The reaction mixtures were separated on a 5% non-denaturing polyacrylamide gel.
Laser scanning confocal fluorescence microscopy
The fluorescence emissions of transformed C.reinhardtii cells were analysed by LSCFM, using a Zeiss LSM 510 META microscopy system (Carl Zeiss, Jena, Germany) based on an Axiovert inverted microscope. cGFP and plastids were excited with the 488 nm line of an argon-ion laser. The fluorescence emission was selected by bandpass filter BP505-530 and longpass filter LP560, respectively, using beam splitters HFT UV/488/543/633 and NFT545.
Sequences, alignments and phylogenetic analysis
Sequences were retrieved from GenBank (NCBI) at http://www.ncbi.nlm.nih.gov, the C.reinhardtii JGI database v2.0 (DOE Joint Genome Institute) at http://genome.jgi-psf.org/chlre2/chlre2.home.html, the Thalassiosira pseudonana JGI database v1.0 at http://genome.jgi-psf.org/thaps1/thaps1.home.html, the Porphyra yezoensis EST index at http://www.kazusa.or.jp/en/plant/porphyra/EST/ (25,26) and the CyanoBase genome database at http://www.kazusa.or.jp/cyano/. Multiple sequence alignments of NAPs were performed using ClustalW software (27). Sequence alignments were displayed with GENEDOC (28) and refined manually. The phylogenetic tree was generated based on trimmed protein sequences with programs contained in the program package PHYLIP version 3.63 (29). An alignment with sequences from mature proteins was impossible since all proteins differ largely in length and some were only available from partial cDNAs (AU191247 , BM448458 ) obtained from EST libraries. Analyses with the PHYLIP package were performed using PROTDIST/NEIGHBOR and the PROTPARS algorithms for distance matrix and maximum parsimony analysis, respectively. Support for the branches was estimated by bootstrap analysis of 2000 replicates generated with SEQBOOT, and a majority rule consensus tree was generated using the program CONSENSE. The resulting tree topology was then visualized in TREEVIEW (30). For analyses of putative targeting signal sequences, ChloroP (31), LOCtree (32), TargetP (33), GENOPLANTETM PREDOTAR (http://www.genoplante.com/) and PSORT (34) were used. Isoelectric point and sequence masses were calculated by the program WinPep 3.01 (35).
RESULTS
Identification of a tscA-specific RNA-binding protein using the yeast three-hybrid system
To identify RNA-binding proteins most probably involved in organellar group II intron splicing, we performed an in vivo screen using the yeast three-hybrid system. In this system, an RNA–protein interaction is detected by the reconstitution of the yeast transcriptional activator GAL4 and subsequent activation of reporter genes. The system is based on one hybrid RNA and two recombinant fusion proteins containing the GAL4 DNA-binding and GAL4 transcription activation domains, respectively. To isolate chloroplast intron RNA-binding proteins of C.reinhardtii, a cDNA library was screened using the yeast three-hybrid system developed by (21). In this system, the chimeric protein RevM10, fused to a gene fragment encoding the DNA-binding domain of GAL4 (GAL4-DB RevM10), binds to RRE, which is part of a chimeric RNA. The 3' part of this RNA consists of the tscA RNA which functions as bait during the screen (RRE-tscA). Chimeric proteins serve as prey and are encoded by cDNA clones of a C.reinhardtii cDNA library. All cDNAs were fused to a gene fragment encoding the transcription activation domain of GAL4 (GAL4-AD). The reliability of this three-hybrid screen was tested with a recombinant RNA carrying two copies of RRE (RRE–RRE), co-expressed with the two RevM10 fusion proteins (AD-RevM10, DB-RevM10). RRE and RevM10 are known to interact with each other (21). As depicted in lane 1 of Figure 1, the interaction resulted in reconstitution of GAL4 activity.
Figure 1 Schematic overview of selected constructs used in the yeast three-hybrid analyses. In each case, five representative yeast transformants were tested for their ability to activate ?-galactosidase by a colony colour assay. Abbreviations: Lhcb3', fragment of 3'-UTR of the Lhcb1 transcript (37); cNAPL, chloroplast NAP-like; GAL4-AD, activation domain of GAL4 transcription factor; GAL4-DB, DNA-binding domain of GAL4 transcription factor; psaADV+VI, domains DV and DVI of the first psaA intron (4); RevM10, RNA-binding protein RevM10 (21); rI1DIV-VI, domains DIV to DVI of intron rI1 (36); RRE, Rev responsive element that is specifically bound by RevM10 (21); tscA, processed tscA RNA (6).
For a global C.reinhardtii cDNA screen, the yeast strain CG1945 was co-transformed with pRevtscA1, expressing both the hybrid protein DB-RevM10 and the recombinant RNA. This strain served as host for transformation of the library of pGAD10-derived plasmids harbouring cDNA sequences of C.reinhardtii. A total of 2.1 x 107 clones were analysed and 14 clones were identified to bind to tscA RNA. Interestingly, of these 14 candidate cDNAs, 13 encoded identical polypeptides with significant homology to NAPs. Because of its binding to chloroplast intron RNA, the NAP-like polypeptide was designated as cNAPL.
To assess the specificity of the three-hybrid interaction between tscA RNA and cNAPL, several controls were carried out. The specificity of the tscA interaction was tested against two further hybrid RNAs. Recombinant control RNAs containing sequences of domains DIV to DVI of the heterologous intron rI1 (36) (RRE-rI1DIV-VI) and a fragment of the 3'-untranslated region (3'-UTR) of the Lhcb1 transcript (37) (RRE-Lhcb3') were fused separately to the RRE sequence, respectively. As shown in lanes 5, 7 and 8 of Figure 1, activity of the reporter ?-galactosidase is dependent on the presence of the RRE-tscA hybrid RNA and the cNAPL-AD fusion protein. These results indicate a specific interaction of cNAPL with the tscA transcript. In addition, we were able to demonstrate that cNAPL also binds to domains DV and DVI of the first psaA intron (RRE-psaADV+VI) as seen in lane 6 (Figure 1). To exclude one- or two-hybrid interaction or unspecific RNA interaction, appropriate negative controls were also performed in the filter assay, including different combinations of the three-hybrid components, represented in lanes 2–4 (Figure 1). In the absence of any one of the hybrid components, transformants displayed no ?-galactosidase activity. Results of the three-hybrid analysis, therefore, clearly demonstrate the specific interaction of cNAPL with tscA RNA and the 3' part of the first intron of psaA RNA.
In vitro binding of cNAPL to chloroplast transcripts
To confirm the specific interaction between cNAPL and tscA RNA in the yeast three-hybrid system, we performed in vitro binding studies using EMSA. For synthesis of cNAPL, the cNapl gene was fused with six histidine residues for expression in E.coli. After purification of the recombinant His-tagged protein with Ni2+-NTA chromatography, cNAPL was incubated with in vitro transcribed radioactively labelled RNAs corresponding to domains DII and DIII of tscA RNA. As shown in Figure 2a, domains DII and DIII range from nt 75 to 218 and from 220 to 400, respectively. Together, they cover nt 75–400. RNAs were incubated in the absence or presence of recombinant cNAPL and fractionated on native gels (Figure 2b–i). In Figure 2b–d, increasing amounts of cNAPL (2–20 μg) were incubated with 30 fmol of in vitro synthesized transcripts of tscA intron RNA fragments comprising either domain DII, domain DIII or both domains DII+III. Arrows indicate RNA–protein complexes as multiple shifted bands that can be observed when the RNA is incubated with increasing amounts of cNAPL polypeptide.
Figure 2 In vitro binding of cNAPL to representative intron domains of tscA and psaA RNA. (a) Secondary structure model of the first psaA intron (6). The tripartite group II intron consists of three separate RNA molecules (tscA, 5' intron exon 1, 3' intron exon 2). Arrows indicate 5'–3' strand polarity of the three separate transcripts. Roman letters denote the six conserved domains of group II introns (DI-DVI). Black numerals correspond to nucleotides of the processed tscA RNA sequence and to the 3' intron sequence of psaA exon 2. The –' tertiary interaction is symbolized by a dashed line (79). The –' interaction is indicated (72). The peripheral structures of domains DI and DIV could not be unambiguously determined and are not represented, modified according to (6). (b–e) RNA EMSAs of domain DII and DIII of tscA RNA and domain DV+VI of the first psaA intron with increasing amounts of cNAPL. EMSAs were performed by incubating 30 fmol internally labelled intron RNA (tscADII, tscADIII, tscADII+III, psaADV+VI) with increasing amounts of affinity-purified cNAPL polypeptide (numbers indicate the amounts of protein in μg) or without protein (0). Affinity-purified CEFEF protein of Acremonium chrysogenum was used as a negative control (C). RNA–protein complexes were resolved from free RNA (F) by electrophoresis in 5 % native polyacrylamide gels. Arrows indicate shifted bands. (f–i) Competition assays of cNAPL using 30 fmol radioactive probes of internally labelled intron RNA (tscADII, tscADIII, tscADII+III, psaADV+VI) and excess of cold specific (tscADII, tscADIII, tscADII+III, psaADV+VI) and non-specific competitors (pBIIKS+). Lanes covered with triangle on top are 0-, 2-, 5- and 10-fold molar excess of competitor, respectively, in the case of tscA RNA and 0-, 2.5-, 5-, 10- and 20-fold molar excess of competitor, respectively, in the case of psaA. Free RNA, negative control and shifted bands are indicated as in (b–e).
To investigate the binding specificity of cNAPL, competition analyses were performed. The above-mentioned radiolabelled RNAs were incubated with a constant amount of 15 μg of cNAPL protein in the presence of increasing amounts of unlabelled specific and non-specific competitor RNAs, respectively. As shown in Figure 2f–h, the presence of just a 2-fold molar excess of unlabelled specific competitor RNA almost completely abolished the formation of a complex between cNAPL and the labelled target tscA RNA. In contrast, the presence of the same amount of non-specific competitor RNA, namely pBIIKS+ (121 nt), had no significant effect on the formation of the complex with the labelled target RNAs. In addition, EMSA was performed with in vitro synthesized RNA of domains DV and DVI of the first psaA intron. As shown in Figure 2a, the two domains range from nt 108 to 192. EMSAs with increasing amounts of cNAPL protein (Figure 2e) and competition analysis (Figure 2i) indicate that cNAPL binds specifically to domains DV and DVI of the first psaA intron, albeit with lower affinity than to tscA RNA.
Chloroplast transcripts are rich in A and U residues. To study the sequence preferences, we compared the ability of A, C, G or U RNA homopolymers to compete for binding of cNAPL to the labelled domain DII of the tscA RNA and domain DV+VI of the first psaA intron, respectively. As depicted in Figure 3a, excess amounts of poly(U) abolished cNAPL binding, while the same amounts of poly(C) or poly(G) have no significant effect on the formation of the complex. Using poly(A) resulted in a weak reduction of the binding specificity. As depicted in Figure 3b, binding of cNAPL to domain DV and DVI of the first psaA intron was also competed by poly(U) and in addition by poly(A) and to a lesser extent by higher amounts of poly(G) (0.2 and 0.5 μg). The competition assays suggest that cNAPL preferentially interacts with U-rich sequences. To further investigate the specificity of cNAPL for the tscA RNA, total wild-type RNA was used as competitor in EMSAs. As shown in Figure 3a and b, 0.5 μg of total RNA competed the binding of cNAPL to the tscA and psaA intron RNA, respectively, suggesting that there are other transcripts bound by cNAPL. Simultaneously, we examined the binding of cNAPL to transcripts of four chloroplast genes and three nuclear genes. As given in Figure 3c, cNAPL binds to 5'-UTRs of chloroplast psbA, psbD, rbcL and rps4 RNA and to a lesser extent to nuclear Lhcb1 5'-UTR and Rps18. No binding is observed to nuclear Lhcb1 3'-UTR and Tba1A. To investigate the binding specificity of cNAPL to the tscA RNA, unlabelled RNA of the above-mentioned chloroplast and nuclear transcripts was used in competition assays. As shown in Figure 3d, a 50-fold molar excess of Lhcb1 3'-UTR, Tba1A and a 15-fold molar excess of Lhcb1 5'-UTR had no significant effect on the formation of the complex, whereas a 50-fold molar excess of psbA 5'-UTR, psbD 5'-UTR and Rps18 resulted in a slight decrease in tscA RNA–protein complex formation. Interestingly, similar results in competition experiments were obtained when binding of cNAPL to the labelled tscA RNA was competed by a 50-fold molar excess of 5'-UTRs of rbcL or rps4, or by a 2-fold molar excess of unlabelled tscA RNA. Thus, cNAPL shows specificity for the tscA RNA and to a lesser extent to other U-rich chloroplast transcripts as well. The overall comparison of all tested in vitro transcripts revealed that binding of cNAPL depends on the U-content and stretches of Us (data not shown).
Figure 3 Binding specificity of cNAPL. (a and b) RNA EMSAs of domain DII+III of tscA RNA (30 fmol) and domain DV+VI of the first psaA intron (40 fmol) with cNAPL in the presence of no competitors (-), the unlabelled RNA homopolymer competitors poly(U), poly(A), poly(C), poly(G) or the unlabelled wt total RNA. Numbers indicate the amount of competitor in μg. (c) Chloroplast transcripts (30 fmol) corresponding to fragments of 5'-UTRs (5'), 3'-UTR (3') and to regions of coding sequences were tested as indicated. (d) Competition assay of cNAPL using 30 fmol of labelled domain DII+III of tscA RNA and excess of cold specific or unspecific competitors as indicated. (e) RNA EMSA of domain DI (20 fmol), DII+III (40 fmol), DIV (60 fmol) and DV+VI (60 fmol) of heterologous intron rI1 of S.obliquus with cNAPL. (f) DNA EMSA of cNAPL using 30 fmol of labelled PCR fragments as indicated. DNA was incubated in the absence (0) or presence (15 μg) of recombinant cNAPL. RNA (30 fmol) serves as a control for shift conditions. RNA in all experiments was incubated in the absence (0) or presence (15 μg) of affinity-purified cNAPL. Free RNA, controls and shifted bands are denoted as in Figure 2b–e.
We further tested whether the typical secondary structures of group II intron domains mediate cNAPL binding. In Figure 3e, only domains with U-rich sequences are able to shift cNAPL, indicating that the structure is not relevant for binding. For example, the U-rich domain DV+VI of the first psaA intron (Figure 2e) show cNAPL binding, while the corresponding domains of the heterologous intron rI1 of Scenedesmus obliquus do not. Furthermore, the experiment in Figure 3f shows that cNAPL do not have DNA-binding activity.
Taken together, these results indicate that cNAPL binds specifically the first psaA group II intron and thus confirm data from the yeast three-hybrid analyses. Furthermore, cNAPL has affinities to U-rich chloroplast 5'-UTRs. Hence, the data imply that the RNA-binding protein cNAPL may play a more general role in the RNA metabolism of C.reinhardtii.
Characterization of a chloroplast NAP-like polypeptide
The nucleotide sequence of the cNapl cDNA (pGAD6-19) consists of 1642 bp with a TGTTA polyadenylation site located 139 bp upstream of the poly(A) tail in the 5'-UTR. The 3'-UTR region is 588 bp in length, and a 5' rapid amplification of cDNA ends (RACE) analysis revealed 22 bp for the 5'-UTR (data not shown). Sequence analysis identified an open reading frame of 1053 bp and 350 amino acids, encoding a polypeptide with a predicted size of 39.4 kDa. The amino acid composition of the protein revealed 20.6% acidic and 13.5% basic residues with an estimated pI of 4.33. The hydropathy profile calculated according to (38) indicates mostly hydrophilic regions, suggesting that cNAPL is a soluble protein. The neural-network based predictors for subcellular localization of proteins ChloroP (31) and LOCtree (32) revealed a putative transit sequence for import into the chloroplast at the N-terminal end of cNAPL, whereas TargetP proposes a mitochondrial localization. However, for chloroplast transit peptides of Chlamydomonas, it was shown that they are more similar to mitochondrial targeting peptides than to chloroplast transit peptides of higher plants (39). The N-terminal amino acid sequence of cNAPL possesses features typical of chloroplast transit peptides, such as an alanine as the second residue. The N-terminal end is an uncharged region with an abundance of arginine (13.6%), valine (13.6%) and alanine (18.2%) residues. This transit peptide could be 46 amino acids long since a possible cleavage site sequence Val-Gly-Ala is present (39). The cleavage would give rise to a predicted mature protein of 304 amino acids with a molecular mass of 34.6 kDa.
Comparison of the genomic and cDNA sequences of cNapl using the C.reinhardtii JGI database (DOE Joint Genome Institute) reveals the presence of 9 large introns and 10 exons (Figure 4). As mentioned above, database searches detected significant sequence similarity with NAPs. Using the C.reinhardtii JGI database, we could also identify a Chlamydomonas homologue of a NAP that does not possess a chloroplast targeting signal. Furthermore, another algal NAP homologue was detected in the marine centric diatom T.pseudonana using the T.pseudonana JGI database (DOE Joint Genome Institute). Figure 5 shows a multiple alignment of cNAPL with NAP polypeptides of algae and higher plants that displayed the highest homology using the BLASTP algorithm (40). Regions of sequence homology between these proteins were found over the entire length of the amino acid sequence, including several blocks of sequence identity. A total of 50 amino acids are identically positioned within all 8 sequences. Overall, there is 29% identity between cNAPL and the other NAP proteins, indicating that cNAPL is a member of the NAP family. With 46% identical amino acids, the Chlamydomonas NAP polypeptide shows a higher degree of homology to plant NAPs than cNAPL. However, Chlamydomonas cNAPL and Thalassiosira NAP share common features atypical for NAPs. They possess, for example, an N-terminal extension that is not found in plant NAPs. In cNAPL, this extension was shown in silico to correspond to a chloroplast targeting signal, whereas an unambiguously prediction is currently impossible for the extension in the sequence of Thalassiosira NAP, because of its complex plastids and multipartite plastid targeting signals (41). Interestingly, no chloroplast transit signal could be identified in all currently available NAP sequences of higher plants.
Figure 4 Physical map of the cNapl gene. Exons (1–10) and introns are represented by closed and open boxes, respectively, with their corresponding size in base pairs. The arrow indicates transcriptional direction. Oligoucleotides used for RT–PCR analysis are given as arrowheads.
When we compared the protein sequences of NAPs from different sources, several evolutionarily conserved domains, such as stretches of acidic residues, as well as nuclear transport signal sequences, were detected (12,42). Recently, Dong and co-worker (42) identified a 16 amino acid sequence in the N-terminal regions of NAP-like sequences from rice and tobacco that is similar to the nuclear export signal (NES) of the yeast NAP1 protein. From the comparison in Figure 5, it is evident that this 16 amino acid stretch is absent in the C.reinhardtii cNAPL sequence. However, there are stretches of amino acid residues in the central and C-terminal part of the sequences of Arabidopsis, maize, pea, rice, soybean and Chlamydomonas NAP that have characteristics of nuclear localization signals (43). The NLS sequences are similar to PKKKKRKV, the NLS found in SV40 T antigen (44). In contrast, cNAPL and Thalassiosira NAP do not possess any NLS sequences. The alignment in Figure 5 reveals two clusters of highly acidic amino acids at the C-terminus of higher plants and Chlamydomonas NAP; both these clusters can also be found in similar positions in the amino acid sequences of yeast NAP1 and HeLa hNRP. Interestingly, however, neither of these clusters is present in cNAPL and Thalassiosira NAP.
Figure 5 Amino acid sequence comparison of C.reinhardtii cNAPL, NAP and NAPs of higher plants and T.pseudonana. Black shading denotes distinct similarities of the tscA-binding protein cNAPL to NAPs. Grey shading shows the higher degree of homology of the NAP protein from C.reinhardtii to NAP proteins of higher plants in contrast to cNAPL. Arrows delimit the NAP domain. A putative prenylation motif is underlined, putative nuclear localization signals are framed and a putative NES is underlined with ‘’. A putative chloroplast targeting signal sequence is represented by ‘+’ on top of the alignment. The two acidic amino acid regions are underlined with dashed lines. An asterisk indicates identical residues. Similar residues are indicated by one or two dots, depending on the degree of similarity of the relevant amino acids. The following protein sequences were used for comparison: At, A.thaliana AAL47354; Cr, C.reinhardtii C_1660026 (JGI); Gm, Glycine max AAA88792; Os, Oryza sativa XP_475795; Ps, Pisum sativum T06807; Tp, T.pseudonana 115707 (JGI); Zm, Zea mays AY100480.
Furthermore, Arabidopsis, maize, pea, rice, soybean and Chlamydomonas NAP contain the CKQQ sequence at their C-terminal ends, a motif suggested as a potential prenylation signal. This signal is supposed to promote membrane interactions and appears to play a major role in several protein–protein interactions. The CKQQ sequence was also identified in NAP polypeptides of rice and tobacco (45). The prenylation signal is missing in cNAPL and Thalassiosira NAP.
To analyse the expression of cNapl in the wild-type strain, RT–PCR analysis was carried out with specific primers designed to amplify cNapl from the start (ATG) to the stop (TAG) codon (Figure 4). As shown in Figure 6a, a single band of around the expected size of 1 kb was obtained. This result indicates the specific expression of cNapl in C.reinhardtii wild-type strain. To examine cNapl expression, total RNA was prepared from wild-type strains CC406 cw15 and arg-cw15 and photosystem I mutants T11-1 59 and T11-3 39 (S. Glanz, unpublished data). Northern blot analysis was carried out using the 32P-labelled 1.7 kb fragment of the cNapl cDNA as probe. As shown in Figure 6b, the probe detected a specific transcript of 1.6 kb, which is consistent with the expected size of the cNapl transcript. An equal load check using Rps18 (46) revealed no difference between transcript accumulation in wild-type versus photosystem I mutant strains. Thus, pleiotropic phenotypes that are often associated with photosynthetic defects do not affect transcriptional expression of the cNapl gene.
Figure 6 Analyses of cNapl transcript. (a) RT–PCR analysis of cNapl transcripts in wild-type arg-cw15. RT–PCR was carried out by using either DNase treated poly(A) RNA as a control (cw15 RNA), cDNA (cw15 cDNA) or genomic DNA (cw15 DNA) as template. Oligonucleotides used for RT–PCR are for_6-19_BspHI and rev_6-19_2xMet_HindIII. Their position is shown in Figure 4. Oligonucleotides rpsF2 and rpsR2 specifically amplify a fragment of the Rps18 transcript and serve as a control for cDNA preparation. Abbreviation: Rps18, cytoplasmic ribosomal protein S18 of C.reinhardtii (46). (b) RNA blot analysis of cNapl transcripts in wild-type and non-photosynthetic mutant strains. Total RNA (20 μg) of wild-type strains CC406 cw15 and arg-cw15 and photosystem I mutants T11-3 39 and T11-1 59 (S. Glanz, unpublished data) were separated on a 1% agarose/formaldehyde gel, transferred onto a nylon membrane and hybridised separately with a cNapl- and Rps18-specific probe as a loading control.
Cellular localization of the cNAPL
The N-terminal 46 amino acids of cNAPL were predicted to be a signal sequence because of certain structural features. To determine the cellular localization of the cNAPL polypeptide, we performed LSCFM. A map of the constructs is shown in Figure 7a. As can be seen in Figure 7b, non-transformed arg-cw15 cells (wt) do not show any fluorescence when green fluorescent protein (GFP) was excited. The complete N-terminal signal sequence (46 amino acids) and the sequence for the first 38 amino acids of the mature cNAPL were fused to a synthetic GFP sequence (47) (N84-cGFP) in frame, keeping the putative N-terminal transit peptide functional. Previous work on diatom protein localization established that the complete pre-sequence of plastid preproteins is sufficient to drive the import of GFP into plastids (41). A similar strategy was successfully applied recently for localization of the phage-type organellar RNA polymerases of Arabidopsis thaliana and Chenopodium album (48). To determine the cellular localization of cNAPL in the algal cell, the cNaplN84-cgfp fusion gene was expressed in C.reinhardtii cells under the control of the HSP70A/RBCS2 promoter (49). As shown in Figure 7b, LSCFM confirmed a plastidic localization of cNAPL. The fluorescent images reveal a co-localization of the chimeric N84-cGFP polypeptide (green fluorescence) with the chloroplast (red autofluorescence of plastids). Right panels show the merged images of cGFP fluorescence and chlorophyll autofluorescence of the same cell. To rule out the possibility that use of the non-native promoter HSP70A/RBCS2 leads to mis-targeting of cNAPL, a gene for a fusion protein with a 31 amino acid deletion in the chloroplast targeting sequence of cNAPLN84 was constructed (N848-39-cGFP). As shown in Figure 7b, this protein clearly localizes in the cytoplasm and to spot-like structures outside of the autofluorescing chloroplast. As further control for nuclear localization, we used cGFP in fusion with the Ble polypeptide (Ble-cGFP). The bacterial ble gene has been successfully expressed in C.reinhardtii by fusing the coding sequence to the 5' and 3' non-coding sequence of the RBCS2 gene, containing additional intron sequences (50). For the Ble polypeptide, it has been shown previously that the bacterial protein is localized in the nucleus (51). This localization was also confirmed by our experiments as demonstrated in Figure 7b and there was no congruence of cGFP fluorescence with the position of the plastid as observed by fluorescence microscopy. In addition, we used the C-terminal cGFP-fused Rps18 polypeptide (Rps18-cGFP) as a marker for cytoplasmic localization. The corresponding cGFP fluorescence signals appeared to accumulate in spots within the cytoplasm. To the best of our knowledge, this is the first demonstration of a cytoplasmic localization of a C.reinhardtii protein using GFP. In conclusion, our results from microscopic investigations clearly demonstrate that the N-terminal extension of cNAPL functions as a chloroplast targeting signal that is able to direct cNAPL to the organelle.
Figure 7 Confocal fluorescence microscopy to demonstrate chloroplast localization of the cNAPL polypeptide. (a) Schematic representation of fusion protein constructs used for cGFP-assays. (b) Laser scanning confocal microscopy of C.reinhardtii arg-cw15 transformants. Transformants were assayed by differential interference contrast microscopy (DIC) or by confocal fluorescence microscopy. DIC, cGFP fluorescence (green) and chlorophyll autofluorescence (red) were merged as indicated. The DIC and cGFP images are representatives of five independent experiments. Scale bar represents 5 μm. Abbreviations: ble, phleomycin resistance gene of Streptoalloteichus hindustanus (50); cgfp, synthetic GFP (47); N84, the N-terminal 84 amino acids of cNAPL; N848-39, the N-terminal 84 amino acids of cNAPL lacking 31 amino acids in the chloroplast targeting sequence; P, HSP70A/RBCS2 promoter (49); Rps18, cytoplasmic ribosomal protein S18 of C.reinhardtii; T, 3'-UTR of Lhcb1 or of RBCS2 gene.
Phylogenetic analyses
The members of the NAP family are considered as histone chaperones, which are conserved from yeast to man. To examine the evolutionary relationships between cNAPL and the above-mentioned homologues, we performed a phylogenetic analysis using the neighbour-joining method. A tree was constructed from multiple alignments of the NAP domains of cNAPL and of NAP polypeptides from various eukaryotes. In addition to the NAP homologue in the diatom T.pseudonana, a partial sequence of a predicted protein with homology to NAPs was found in the EST database of the red alga Porphyra yezoenis (Kazusa DNA Research Institute) (25,26). Similarly, we detected another partial EST homologue (GenBank) from the green alga Dunaliella salina. So far, genomic sequences of these algae are not available yet. For prokaryotes, however, we were unable to detect any NAP or cNAPL homologues in the publicly available genome sequences of bacteria (GenBank) and cyanobacteria (CyanoBase).
The unrooted tree in Figure 8 shows four main clusters: animals, algae, fungi and higher plants. Trees generated with maximum parsimony and Bayesian analysis (52) had an overall similar topography (data not shown). From Figure 8, it becomes evident that the Chlamydomonas NAP polypeptide is related to the group of higher plant NAP proteins, which all lack a chloroplast targeting sequence, whereas cNAPL clusters together with the algal NAP homologues. For the NAP domain, the amino acid sequence of Chlamydomonas NAP shows 54% identity to higher plant NAPs, whereas the NAP domain of cNAPL exhibits 35% identity to higher plant NAPs. The corresponding values for the algal NAPs are 35% for Thalassiosira NAP, 26% for Dunaliella NAP and 15% for Porphyra NAP. Overall, we propose that cNAPL with its chloroplast transit peptide represents a new type of NAP-like polypeptide in photoautotrophic organisms.
Figure 8 Phylogenetic tree of 31 NAP/NAP-like proteins. The tree was generated using PHYLIP (29), as indicated in Material and Methods. Numbers at branches indicate bootstrap support (2000 bootstrap replicates) in percent. Abbreviations: A.f., Aspergillus fumigatus XP_754077; A.t.1, A.thaliana AAL47354; A.t.2, A.thaliana BAA97025; A.t.3, A.thaliana NP_194341; C.n., Cryptococus neoformans AAW43807; D.d., Dictyostelium discoideum EAL71995; D.m., Drosophila melanogaster AAB07898; D.s., D.salina BM448458; G.m., G.max AAA88792; H.s.1, Homo sapiens BAA08904; H.s.2, H.sapiens NP_004528; M.g., Magnaporthe grisea AAX076; N.c., Neurospora crassa CAD70974; N.t.1, Nicotiana tabacum CAD27460; N.t.2, N.tabacum CAD27461; N.t.3, N.tabacum CAD27462; O.s.1, O.sativa AAV88624; O.s.2, O.sativa CAD27459; O.s.3, O.sativa P0456F08; O.s.4, O.sativa XP_475795; P.c., Paramecium caudatum BAB79235; P.s., P.sativum T06807; P.y., P.yezoensis AU191247; R.n.1, Rattus norvegicus AAC67388; R.n.2, R.norvegicus NP_446013; S.c., S.cerevisiae P25293; S.p., Schizzosaccharomyces pombe T41330; T.b., Trypanosoma brucei XP_826968; T.p., T.pseudonana 115707 (JGI).
DISCUSSION
cNAPL is an tscA-specific RNA-binding protein
The splicing chemistry and RNA structures of self-splicing group II introns and nuclear pre-mRNA introns are strikingly similar. Thereby, suggesting some evolutionary relationship between group II introns and the nuclear spliceosomal intron (53). Moreover, there is compelling evidence to suggest that chloroplast spliceosomes catalyse intron splicing using a similar mechanism to that reported for nuclear spliceosomes. This suggestion is the result of data obtained form a combination of different experimental approaches that have identified components of a putative chloroplast spliceosome in C.reinhardtii (7–11) and higher plants (54). Additional support comes from studies with tobacco chloroplasts. Nakamura and co-workers (55) identified chloroplast ribonucleoproteins (cpRNPs) that are associated in vivo with various species of chloroplast mRNAs and intron-containing precursor tRNAs. They suggested that these stromal RNA–protein complexes promote, for example, RNA splicing, processing or editing. Previously, the yeast three-hybrid system was used to demonstrate specific binding of proteins to organellar intron RNA (8,56). In this work, we used the yeast three-hybrid system successfully to screen a C.reinhardtii cDNA library and we were able to detect the novel organellar protein cNAPL that specifically binds to the first psaA group II intron. The binding of cNAPL to domains DII+III and DV+VI of the first psaA intron was further shown in electromobility shift assays.
In several other studies using defined intron domains, separated molecules were found to fold into a structure corresponding to their conformation in the complete active intronic RNA (57,58). Recent studies have demonstrated that fragmented intron molecules are appropriate targets for gel retardation assays (22). In EMSA, we observed multiple shifted bands after incubating domains DII+III of tscA RNA with increasing amounts of cNAPL. As was shown for example for the mutated SxlN1 protein of Drosophila, multiple shifted bands probably correspond to the sequential filling of binding sites when protein concentration is increased (59). Hence, the two shifted bands observed in Figure 2b–d shows non-cooperative binding of cNAPL to domains DII and DIII, thus indicating at least two binding sites. However, we were not able to detect a known RNA-binding motif in cNAPL, such as the RNP motif, the K homology motif (KH) or the RGG (Arg-Gly-Gly) box. Further binding studies with truncated cNAPL constructs could help to determine the exact RNA-binding domain.
cNAPL is phylogenetically related to nuclear localized NAPs and most probably has not been acquired from a cyanobacterial endosymbiont
cNAPL is a member of the multifunctional family of NAPs that are involved in processes which normally take place in the nucleus or the cytoplasm of various eukaryotes (12,42,61). Besides their distinct functions in mitotic events and cytokinesis, NAPs act as histone chaperones, binding to all core histones with a preference for H2A and H2B and working as a deposition factor by transferring NAP1-bound histones to double-stranded DNA. In addition to its nucleosome assembly and histone-binding activity, NAP1 as well as plant NAP1-like proteins may be involved in regulating gene expression and therefore cellular differentiation (45,62,63). NAP1 has also been shown to facilitate transcription factor binding by disruption of the histone octamer through the binding of H2A and H2B (13,64). NAP1 also shuttles histones H2A and H2B as well as a complex of H2A, H2B, H3 and H4 between the template DNA and nascent RNA during transcription (65). To date, however, it is still unknown whether NAP1 interacts directly also with RNA.
Phylogenetic analysis indicates that cNAPL is related to nuclear localized NAP polypeptides. The clustering of cNAPL with algal NAPs and not with NAPs of higher plants could be explained by its chloroplast targeting signal. In the case of Thalassiosira NAP, TargetP predicted a 69 amino acid signal sequence, which could not be assigned clearly to any organelle. Diatoms, such as T.pseudonana, possess so-called ‘complex plastids’ delineated by four distinct membranes. For a nuclear-encoded plastid polypeptide to be directed to the plastids, multiple targeting signals are necessary. Thus, determining a protein's localization in diatoms using today's prediction programs is difficult.
It is now generally accepted that double-membrane-bound chloroplasts are the result of an endosymbiotic event involving a cyanobacterial-like organism early on in evolution. During evolution, the cyanobacterial endosymbiont has lost its autonomy, and this has been accompanied by significant changes of the chloroplast proteome (14,66). A key factor in this process of adoption was the loss of genetic material resulting from gene transfer to the cell nucleus. The vast majority of proteins in present day chloroplasts are encoded by the host nucleus and require N-terminal presequences that target them back to the chloroplast. Therefore, in order to establish a functional organelle–nucleus interaction, chloroplasts have had to import a set of new proteins to adapt their metabolism to the new conditions. Interestingly, a proportion of these proteins do not seem to have been acquired from cyanobacteria (14). Phylogenetic analysis and database research revealed that no NAP homologues could be identified in prokaryotic organisms. We therefore suggest that cNAPL does not originate from an endosymbiotic gene transfer event and that the chloroplast of C.reinhardtii has gained cNAPL as a novel nuclear-encoded factor. The phylogenetic analysis suggests that cNapl originate from a nuclear Nap gene, and is probably the result of a gene duplication event that occurred after separation of higher plants from green algae. We thus propose that cNAPL with its chloroplast transit peptide represents a new type of NAP-like polypeptide.
Is cNAPL a multifunctional protein?
Many group II introns of plants have lost their self-splicing activity during evolution and thus, recruited host-encoded proteins as splicing factors (1,67). The participation of nuclear-encoded proteins in chloroplast splicing could provide a means to control the biogenesis of the photosynthetic apparatus. It has been suggested that these recruited host-encoded proteins probably had other functions in the cell. Many of these proteins show some degree of homology to proteins involved in RNA metabolism. For example, Raa2 of C.reinhardtii shows homology to pseudouridine synthetase; however, such enzymatic activity is not required for trans-splicing of psaA RNA (7). Another example is Mss116p of Saccharomyces cerevisiae, which influences the splicing of all nine group I and all four group II introns in mitochondria (68). Mss116p is related to DEAD-box proteins with RNA chaperone function and may facilitate splicing by resolving misfolded introns. However, it is generally discussed that proteins involved in splicing and interacting with intron RNAs seem to support RNA folding or to stabilize the active conformation, whereas the catalytic potential is clearly located in the RNA itself (69). Furthermore, Mss116p is a bifunctional protein that influences, in addition to splicing of group I and group II introns, some RNA end-processing reactions and translation of a subset of mitochondrial mRNAs. Indeed, for several nuclear-encoded host proteins, it is known that they have additional cellular functions and seem to have been recruited for splicing during evolution (1). Therefore, it has been questioned whether cNAPL is also a multifunctional protein. The analysed interactions of cNAPL with tscA RNA and domains DV and DVI of the first psaA intron implicate a role for cNAPL as a potential tscA RNA processing factor and also as a splicing factor of the first psaA intron. An example for such a bifunctional protein comes from recent work where Rat1 is required for both processing of tscA RNA as well as for splicing of the first psaA intron (8). Another factor involved in tscA RNA processing and splicing of both introns is Raa1-L137H (9), allelic to HN31 (70).
cNAPL binds specifically to domains DII and DVI of the first psaA intron. Domain DII is a phylogenetically less-conserved region and was assigned as functionally unimportant because of its high degree of sequence and structure variations (71). However, Chanfreau and Jacquier (72) could show that the so-called tertiary –' interaction (see also Figure 2a) between DII and DVI of group IIA introns is responsible for a conformational change between the two catalytic transesterification steps. Interestingly, only a few of the known group IIB introns can potentially form this interaction. It is discussed that the majority of the group IIB introns interact with protein factors to change the conformation between the first and the second step (69). The fact that cNAPL interacts with both domains, DII and DVI, implies a role for cNAPL in this conformational change.
Our experiments further demonstrated the binding of cNAPL to domain DV of the first psaA intron. Domain DV is the phylogenetically most conserved sequence and essential for the catalytic splicing reaction. DV shows numerous important tertiary interactions and contains important conserved sequence motifs (69). Overall, we assume that cNAPL presumably has a function in the stabilization and correct folding of the catalytic core of the first psaA intron by interacting with tscA RNA and domains DV and DVI.
In addition, our analyses showed that the binding of cNAPL to tscA RNA was competed efficiently by poly(U) in contrast to other RNA homopolymers. Moreover, cNAPL binds the 5'-UTR of U-rich chloroplast transcripts psbA, psbD, rbcL and rps4 considerably better than the nuclear transcripts Lhcb1 5'-UTR, Lhcb1 3'-UTR, Rps18 and Tba1A. It is proven that the 5'-UTRs of chloroplast transcripts contain the cis-acting determinants for the stabilization of plastid transcripts and are essential for initiation of chloroplast translation by binding trans-acting factors (mostly encoded by nuclear genes) and by interaction with the ribosomal preinitiation complex (73). For example, the stability of the psbD mRNA depends on a nucleus-encoded TPR protein, which is part of a high-molecular weight complex mediating its function via the psbD 5'-UTR (74). For the psbA mRNA it is assumed that a multiprotein complex specifically regulates translation of this mRNA through interaction with a U-rich sequence in the 5'-UTR (75). Furthermore, there are several proteins in Chlamydomonas binding to 5'-UTRs of chloroplast mRNAs, e.g. to 5'-UTRs of atpB, rbcL, rps7 and rps12, that so far have not been characterized and are discussed to be required for translational regulation (76,77). However, the cpRNP complexes seem to be not only involved in regulation of translation, but also in distinct steps of post-transcriptional gene expression, e.g. by facilitating proper RNA folding for intron-containing RNA (78). Recently it was demonstrated that there are two RNP complexes in C.reinhardtii that are involved in trans-splicing of psaA, one in the soluble fraction of the chloroplast and the other one associated with chloroplast membranes, which could provide a link to translation (11).
In conclusion, we have isolated a novel type of group II intron RNA-binding protein using the yeast three-hybrid system. Phylogenetic analysis indicates that the chloroplast located cNAPL protein is encoded by a nuclear gene that probably has evolved from duplication of an ancestral Nap gene. Moreover, it is proposed that cpRNP complexes are global mediators of chloroplast RNA metabolism, which connect transcription and translation in the chloroplast (78). Because of cNAPL's binding to U-rich sequences of chloroplast 5'-UTRs, a more general role for this RNA-binding protein is conceivable, e.g. in translation initiation of several chloroplast transcripts. Thus, cNAPL might represent a component of the cpRNP complex.
ACKNOWLEDGEMENTS
We wish to express our thanks to Profs. Peter Hegemann (Berlin), Christoph Beck (Freiburg) and Dr Ulrich Putz (Hamburg) for the gift of plasmids and Drs Birgit Hoff and Stephan Pollmann for advice in LSCFM and Patricia Cebula and Franziska Thorwirth for help with some experiments. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB480 B3). Funding to pay the Open Access publication charges for this article was provided by DFG (Bonn, Bad Godesberg, Germany).
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*To whom correspondence should be addressed. Tel: +49 234 3226212; Fax: +49 234 3214184; Email: ulrich.kueck@ruhr-uni-bochum.de
ABSTRACT
In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.
INTRODUCTION
Group II introns have been detected in eukaryotic organelles as well as in eubacterial genomes as intervening sequences of protein, tRNA or rRNA coding genes. Common to all group II introns is a conserved secondary structure that consists of six double-helical domains (DI-DVI) radiating from a central wheel (1). Group II introns splice via two sequential transesterifications that in vitro occur in some cases autocatalytically. In vivo, however, splicing is dependent on protein co-factors as was shown by mutant analyses (2,3).
In Chlamydomonas reinhardtii chloroplasts, two trans-splicing introns were found in the psaA gene encoding the major P700 chlorophyll a/b-binding protein (4). In the case of the first intron of the psaA gene, three independently transcribed RNAs associate via tertiary interactions to form a functional group II intron, resulting in trans-splicing of the flanking exon 1 and exon 2 (5). Part of this tripartite group II intron is the tscA RNA that is processed from a chloroplast-encoded precursor RNA. Secondary structure predictions revealed that tscA contains domain DII and DIII as well as partial domains DI and DIV of the conserved secondary group II intron structure (6).
Mutant work in C.reinhardtii has shown that >14 nuclear genes affect the chloroplast trans-splicing reaction, and complementation of some of the characterized mutants led to the identification of the molecular determinants. Although some of the identified polypeptides are related to proteins involved in nucleic acid metabolisms, this function seems to be non-essential for intron splicing (7,8). Similar to nuclear mRNA splicing, these protein components might be part of a chloroplast splicing complex, because they were found in large stromal or membrane-bound RNA-containing complexes (9–11).
To define further components of a putative ‘chloroplast spliceosome’ in C.reinhardtii, we used the tscA RNA as bait to identify novel intron RNA-binding proteins. Using the yeast three-hybrid system, we isolated a polypeptide that seems to be a novel member of the multifunctional nucleosome assembly protein (NAP) family. These well-conserved eukaryotic histone chaperones facilitate, for example, the nucleosome assembly and remodelling of chromatin, and are implicated in transcriptional regulation and cell cycle regulation (12,13). Here, we assign a novel function to a NAP-like protein. Laser scanning confocal fluorescence microscopy (LSCFM) and in vitro binding assays demonstrate that chloroplasts of C.reinhardtii contain a NAP-like protein that specifically binds to organellar group II intron RNA and U-rich chloroplast transcripts. Our data further support the view that during evolution, chloroplasts have acquired novel nuclear components that most probably were not delivered by endosymbiont gene transfer (14).
MATERIALS AND METHODS
Strains, culture conditions and transformation
C.reinhardtii cell wall depleted, arginine auxotroph strain argb-ecw15 as well as wild-type strain CC406 cw15 mt- were obtained from the Chlamydomonas Center (Duke University, Durham, NC, USA) and were grown as described on Tris–acetate–phosphate (TAP) medium (15). When required, the medium was supplemented with 50 μg arginine per ml. Nuclear transformation was carried out using the glass bead method (16) and was performed according to (8) with the following modifications. A total of 1–3 x 106 cells/ml were incubated with 1–5 μg supercoiled or SpeI digested pMF59 (17) plasmid DNA. After transformation, cells were spread on solid TAP medium and incubated in continuous light at 25°C. For selection of transformants carrying pMF59, medium contained 5 μg/ml of zeocin (Invitrogen, Karlsruhe, Germany).
Recombinant plasmids
All recombinant plasmids used for in vitro transcription, PCR analysis, protein synthesis, yeast three-hybrid screening, hybridization or generation of transgenic algal strains are listed in Table 1. Sequences of all oligonucleotides are given in Table 2.
Table 1 Plasmids used in this work
Table 2 Oligonucleotide primer pairs used in PCR and RT–PCR experiments
Molecular biological techniques
Procedures for standard molecular techniques were performed as described previously (18). Escherichia coli strain XL1-blue MRF' served as host for general plasmid construction and maintenance (19). The C.reinhardtii cDNA library contains double-stranded cDNA fragments with EcoRI adaptors cloned into the EcoRI site of pGAD10 in DH10B (BD Biosciences Clontech, Heidelberg, Germany) (20). pGAD10 derivatives were used in the yeast three-hybrid screen. C.reinhardtii total RNA was prepared according to (4) and RNA blot experiments were carried out according to (8). Northern analyses were performed with a radioactively labelled 1690 bp pGAD6-19/EcoRI cNapl-specific probe and a 460 bp pGAD-Rps18/EcoRI–BamHI Rps18-specific probe. cDNA was prepared using OmniscriptTM Reverse Trancriptase (Qiagen, Hilden, Germany) and purified with NucAwayTM Spin Columns (Ambion, Austin, TX, USA) according to the manufacturer's protocols. For PCRs, Taq polymerase (Eppendorf, Hamburg, Germany), Hot MasterTM Taq DNA Polymerase (Eppendorf) and the Expand Long Template PCR System (Roche, Mannheim, Germany) were used. Oligonucleotide primers applied for conventional PCR are listed in Table 2.
The yeast three-hybrid system
The yeast three-hybrid analysis was performed as described previously (8). Plasmids are listed in Table 1. Plasmids pADRevM10 and pRevR2 were generous gifts from Dr U. Putz (University of Hamburg) (21). Plasmid pRevtscA1 (8) was used to screen a C.reinhardtii cDNA library (Table 1).
Electrophoretic mobility shift assays (EMSAs)
Recombinant plasmid pQE70-cNAPL (see above) was used for synthesis of cNAPL in E.coli. Recombinant His-tagged cNAPL protein was purified from E.coli with Ni2+-NTA-agarose resin according to the manufacturer's protocols (Qiagen). For RNA mobility shift assays, uniformly 32P-UTP-labelled run-off transcripts served as substrate RNAs and were generated by in vitro transcription of plasmids as given in Table 1. In vitro transcription and EMSAs were performed as previously reported (22,23). Unlabelled competitor RNAs and non-specific competitor RNA, derived from plasmid pBIIKS+, were synthesized as described (22). DNA mobility shift assays were done essentially as described previously (24) with the following modifications. The probes were amplified by PCR with appropriate plasmid DNA or genomic DNA of strain arg-cw15 in the presence of dATP. The labelled probes were purified by denaturing PAGE. Binding reactions were carried out by incubating 30 fmol of the probes with 15 μg of recombinant cNAPL. The reaction mixtures were separated on a 5% non-denaturing polyacrylamide gel.
Laser scanning confocal fluorescence microscopy
The fluorescence emissions of transformed C.reinhardtii cells were analysed by LSCFM, using a Zeiss LSM 510 META microscopy system (Carl Zeiss, Jena, Germany) based on an Axiovert inverted microscope. cGFP and plastids were excited with the 488 nm line of an argon-ion laser. The fluorescence emission was selected by bandpass filter BP505-530 and longpass filter LP560, respectively, using beam splitters HFT UV/488/543/633 and NFT545.
Sequences, alignments and phylogenetic analysis
Sequences were retrieved from GenBank (NCBI) at http://www.ncbi.nlm.nih.gov, the C.reinhardtii JGI database v2.0 (DOE Joint Genome Institute) at http://genome.jgi-psf.org/chlre2/chlre2.home.html, the Thalassiosira pseudonana JGI database v1.0 at http://genome.jgi-psf.org/thaps1/thaps1.home.html, the Porphyra yezoensis EST index at http://www.kazusa.or.jp/en/plant/porphyra/EST/ (25,26) and the CyanoBase genome database at http://www.kazusa.or.jp/cyano/. Multiple sequence alignments of NAPs were performed using ClustalW software (27). Sequence alignments were displayed with GENEDOC (28) and refined manually. The phylogenetic tree was generated based on trimmed protein sequences with programs contained in the program package PHYLIP version 3.63 (29). An alignment with sequences from mature proteins was impossible since all proteins differ largely in length and some were only available from partial cDNAs (AU191247 , BM448458 ) obtained from EST libraries. Analyses with the PHYLIP package were performed using PROTDIST/NEIGHBOR and the PROTPARS algorithms for distance matrix and maximum parsimony analysis, respectively. Support for the branches was estimated by bootstrap analysis of 2000 replicates generated with SEQBOOT, and a majority rule consensus tree was generated using the program CONSENSE. The resulting tree topology was then visualized in TREEVIEW (30). For analyses of putative targeting signal sequences, ChloroP (31), LOCtree (32), TargetP (33), GENOPLANTETM PREDOTAR (http://www.genoplante.com/) and PSORT (34) were used. Isoelectric point and sequence masses were calculated by the program WinPep 3.01 (35).
RESULTS
Identification of a tscA-specific RNA-binding protein using the yeast three-hybrid system
To identify RNA-binding proteins most probably involved in organellar group II intron splicing, we performed an in vivo screen using the yeast three-hybrid system. In this system, an RNA–protein interaction is detected by the reconstitution of the yeast transcriptional activator GAL4 and subsequent activation of reporter genes. The system is based on one hybrid RNA and two recombinant fusion proteins containing the GAL4 DNA-binding and GAL4 transcription activation domains, respectively. To isolate chloroplast intron RNA-binding proteins of C.reinhardtii, a cDNA library was screened using the yeast three-hybrid system developed by (21). In this system, the chimeric protein RevM10, fused to a gene fragment encoding the DNA-binding domain of GAL4 (GAL4-DB RevM10), binds to RRE, which is part of a chimeric RNA. The 3' part of this RNA consists of the tscA RNA which functions as bait during the screen (RRE-tscA). Chimeric proteins serve as prey and are encoded by cDNA clones of a C.reinhardtii cDNA library. All cDNAs were fused to a gene fragment encoding the transcription activation domain of GAL4 (GAL4-AD). The reliability of this three-hybrid screen was tested with a recombinant RNA carrying two copies of RRE (RRE–RRE), co-expressed with the two RevM10 fusion proteins (AD-RevM10, DB-RevM10). RRE and RevM10 are known to interact with each other (21). As depicted in lane 1 of Figure 1, the interaction resulted in reconstitution of GAL4 activity.
Figure 1 Schematic overview of selected constructs used in the yeast three-hybrid analyses. In each case, five representative yeast transformants were tested for their ability to activate ?-galactosidase by a colony colour assay. Abbreviations: Lhcb3', fragment of 3'-UTR of the Lhcb1 transcript (37); cNAPL, chloroplast NAP-like; GAL4-AD, activation domain of GAL4 transcription factor; GAL4-DB, DNA-binding domain of GAL4 transcription factor; psaADV+VI, domains DV and DVI of the first psaA intron (4); RevM10, RNA-binding protein RevM10 (21); rI1DIV-VI, domains DIV to DVI of intron rI1 (36); RRE, Rev responsive element that is specifically bound by RevM10 (21); tscA, processed tscA RNA (6).
For a global C.reinhardtii cDNA screen, the yeast strain CG1945 was co-transformed with pRevtscA1, expressing both the hybrid protein DB-RevM10 and the recombinant RNA. This strain served as host for transformation of the library of pGAD10-derived plasmids harbouring cDNA sequences of C.reinhardtii. A total of 2.1 x 107 clones were analysed and 14 clones were identified to bind to tscA RNA. Interestingly, of these 14 candidate cDNAs, 13 encoded identical polypeptides with significant homology to NAPs. Because of its binding to chloroplast intron RNA, the NAP-like polypeptide was designated as cNAPL.
To assess the specificity of the three-hybrid interaction between tscA RNA and cNAPL, several controls were carried out. The specificity of the tscA interaction was tested against two further hybrid RNAs. Recombinant control RNAs containing sequences of domains DIV to DVI of the heterologous intron rI1 (36) (RRE-rI1DIV-VI) and a fragment of the 3'-untranslated region (3'-UTR) of the Lhcb1 transcript (37) (RRE-Lhcb3') were fused separately to the RRE sequence, respectively. As shown in lanes 5, 7 and 8 of Figure 1, activity of the reporter ?-galactosidase is dependent on the presence of the RRE-tscA hybrid RNA and the cNAPL-AD fusion protein. These results indicate a specific interaction of cNAPL with the tscA transcript. In addition, we were able to demonstrate that cNAPL also binds to domains DV and DVI of the first psaA intron (RRE-psaADV+VI) as seen in lane 6 (Figure 1). To exclude one- or two-hybrid interaction or unspecific RNA interaction, appropriate negative controls were also performed in the filter assay, including different combinations of the three-hybrid components, represented in lanes 2–4 (Figure 1). In the absence of any one of the hybrid components, transformants displayed no ?-galactosidase activity. Results of the three-hybrid analysis, therefore, clearly demonstrate the specific interaction of cNAPL with tscA RNA and the 3' part of the first intron of psaA RNA.
In vitro binding of cNAPL to chloroplast transcripts
To confirm the specific interaction between cNAPL and tscA RNA in the yeast three-hybrid system, we performed in vitro binding studies using EMSA. For synthesis of cNAPL, the cNapl gene was fused with six histidine residues for expression in E.coli. After purification of the recombinant His-tagged protein with Ni2+-NTA chromatography, cNAPL was incubated with in vitro transcribed radioactively labelled RNAs corresponding to domains DII and DIII of tscA RNA. As shown in Figure 2a, domains DII and DIII range from nt 75 to 218 and from 220 to 400, respectively. Together, they cover nt 75–400. RNAs were incubated in the absence or presence of recombinant cNAPL and fractionated on native gels (Figure 2b–i). In Figure 2b–d, increasing amounts of cNAPL (2–20 μg) were incubated with 30 fmol of in vitro synthesized transcripts of tscA intron RNA fragments comprising either domain DII, domain DIII or both domains DII+III. Arrows indicate RNA–protein complexes as multiple shifted bands that can be observed when the RNA is incubated with increasing amounts of cNAPL polypeptide.
Figure 2 In vitro binding of cNAPL to representative intron domains of tscA and psaA RNA. (a) Secondary structure model of the first psaA intron (6). The tripartite group II intron consists of three separate RNA molecules (tscA, 5' intron exon 1, 3' intron exon 2). Arrows indicate 5'–3' strand polarity of the three separate transcripts. Roman letters denote the six conserved domains of group II introns (DI-DVI). Black numerals correspond to nucleotides of the processed tscA RNA sequence and to the 3' intron sequence of psaA exon 2. The –' tertiary interaction is symbolized by a dashed line (79). The –' interaction is indicated (72). The peripheral structures of domains DI and DIV could not be unambiguously determined and are not represented, modified according to (6). (b–e) RNA EMSAs of domain DII and DIII of tscA RNA and domain DV+VI of the first psaA intron with increasing amounts of cNAPL. EMSAs were performed by incubating 30 fmol internally labelled intron RNA (tscADII, tscADIII, tscADII+III, psaADV+VI) with increasing amounts of affinity-purified cNAPL polypeptide (numbers indicate the amounts of protein in μg) or without protein (0). Affinity-purified CEFEF protein of Acremonium chrysogenum was used as a negative control (C). RNA–protein complexes were resolved from free RNA (F) by electrophoresis in 5 % native polyacrylamide gels. Arrows indicate shifted bands. (f–i) Competition assays of cNAPL using 30 fmol radioactive probes of internally labelled intron RNA (tscADII, tscADIII, tscADII+III, psaADV+VI) and excess of cold specific (tscADII, tscADIII, tscADII+III, psaADV+VI) and non-specific competitors (pBIIKS+). Lanes covered with triangle on top are 0-, 2-, 5- and 10-fold molar excess of competitor, respectively, in the case of tscA RNA and 0-, 2.5-, 5-, 10- and 20-fold molar excess of competitor, respectively, in the case of psaA. Free RNA, negative control and shifted bands are indicated as in (b–e).
To investigate the binding specificity of cNAPL, competition analyses were performed. The above-mentioned radiolabelled RNAs were incubated with a constant amount of 15 μg of cNAPL protein in the presence of increasing amounts of unlabelled specific and non-specific competitor RNAs, respectively. As shown in Figure 2f–h, the presence of just a 2-fold molar excess of unlabelled specific competitor RNA almost completely abolished the formation of a complex between cNAPL and the labelled target tscA RNA. In contrast, the presence of the same amount of non-specific competitor RNA, namely pBIIKS+ (121 nt), had no significant effect on the formation of the complex with the labelled target RNAs. In addition, EMSA was performed with in vitro synthesized RNA of domains DV and DVI of the first psaA intron. As shown in Figure 2a, the two domains range from nt 108 to 192. EMSAs with increasing amounts of cNAPL protein (Figure 2e) and competition analysis (Figure 2i) indicate that cNAPL binds specifically to domains DV and DVI of the first psaA intron, albeit with lower affinity than to tscA RNA.
Chloroplast transcripts are rich in A and U residues. To study the sequence preferences, we compared the ability of A, C, G or U RNA homopolymers to compete for binding of cNAPL to the labelled domain DII of the tscA RNA and domain DV+VI of the first psaA intron, respectively. As depicted in Figure 3a, excess amounts of poly(U) abolished cNAPL binding, while the same amounts of poly(C) or poly(G) have no significant effect on the formation of the complex. Using poly(A) resulted in a weak reduction of the binding specificity. As depicted in Figure 3b, binding of cNAPL to domain DV and DVI of the first psaA intron was also competed by poly(U) and in addition by poly(A) and to a lesser extent by higher amounts of poly(G) (0.2 and 0.5 μg). The competition assays suggest that cNAPL preferentially interacts with U-rich sequences. To further investigate the specificity of cNAPL for the tscA RNA, total wild-type RNA was used as competitor in EMSAs. As shown in Figure 3a and b, 0.5 μg of total RNA competed the binding of cNAPL to the tscA and psaA intron RNA, respectively, suggesting that there are other transcripts bound by cNAPL. Simultaneously, we examined the binding of cNAPL to transcripts of four chloroplast genes and three nuclear genes. As given in Figure 3c, cNAPL binds to 5'-UTRs of chloroplast psbA, psbD, rbcL and rps4 RNA and to a lesser extent to nuclear Lhcb1 5'-UTR and Rps18. No binding is observed to nuclear Lhcb1 3'-UTR and Tba1A. To investigate the binding specificity of cNAPL to the tscA RNA, unlabelled RNA of the above-mentioned chloroplast and nuclear transcripts was used in competition assays. As shown in Figure 3d, a 50-fold molar excess of Lhcb1 3'-UTR, Tba1A and a 15-fold molar excess of Lhcb1 5'-UTR had no significant effect on the formation of the complex, whereas a 50-fold molar excess of psbA 5'-UTR, psbD 5'-UTR and Rps18 resulted in a slight decrease in tscA RNA–protein complex formation. Interestingly, similar results in competition experiments were obtained when binding of cNAPL to the labelled tscA RNA was competed by a 50-fold molar excess of 5'-UTRs of rbcL or rps4, or by a 2-fold molar excess of unlabelled tscA RNA. Thus, cNAPL shows specificity for the tscA RNA and to a lesser extent to other U-rich chloroplast transcripts as well. The overall comparison of all tested in vitro transcripts revealed that binding of cNAPL depends on the U-content and stretches of Us (data not shown).
Figure 3 Binding specificity of cNAPL. (a and b) RNA EMSAs of domain DII+III of tscA RNA (30 fmol) and domain DV+VI of the first psaA intron (40 fmol) with cNAPL in the presence of no competitors (-), the unlabelled RNA homopolymer competitors poly(U), poly(A), poly(C), poly(G) or the unlabelled wt total RNA. Numbers indicate the amount of competitor in μg. (c) Chloroplast transcripts (30 fmol) corresponding to fragments of 5'-UTRs (5'), 3'-UTR (3') and to regions of coding sequences were tested as indicated. (d) Competition assay of cNAPL using 30 fmol of labelled domain DII+III of tscA RNA and excess of cold specific or unspecific competitors as indicated. (e) RNA EMSA of domain DI (20 fmol), DII+III (40 fmol), DIV (60 fmol) and DV+VI (60 fmol) of heterologous intron rI1 of S.obliquus with cNAPL. (f) DNA EMSA of cNAPL using 30 fmol of labelled PCR fragments as indicated. DNA was incubated in the absence (0) or presence (15 μg) of recombinant cNAPL. RNA (30 fmol) serves as a control for shift conditions. RNA in all experiments was incubated in the absence (0) or presence (15 μg) of affinity-purified cNAPL. Free RNA, controls and shifted bands are denoted as in Figure 2b–e.
We further tested whether the typical secondary structures of group II intron domains mediate cNAPL binding. In Figure 3e, only domains with U-rich sequences are able to shift cNAPL, indicating that the structure is not relevant for binding. For example, the U-rich domain DV+VI of the first psaA intron (Figure 2e) show cNAPL binding, while the corresponding domains of the heterologous intron rI1 of Scenedesmus obliquus do not. Furthermore, the experiment in Figure 3f shows that cNAPL do not have DNA-binding activity.
Taken together, these results indicate that cNAPL binds specifically the first psaA group II intron and thus confirm data from the yeast three-hybrid analyses. Furthermore, cNAPL has affinities to U-rich chloroplast 5'-UTRs. Hence, the data imply that the RNA-binding protein cNAPL may play a more general role in the RNA metabolism of C.reinhardtii.
Characterization of a chloroplast NAP-like polypeptide
The nucleotide sequence of the cNapl cDNA (pGAD6-19) consists of 1642 bp with a TGTTA polyadenylation site located 139 bp upstream of the poly(A) tail in the 5'-UTR. The 3'-UTR region is 588 bp in length, and a 5' rapid amplification of cDNA ends (RACE) analysis revealed 22 bp for the 5'-UTR (data not shown). Sequence analysis identified an open reading frame of 1053 bp and 350 amino acids, encoding a polypeptide with a predicted size of 39.4 kDa. The amino acid composition of the protein revealed 20.6% acidic and 13.5% basic residues with an estimated pI of 4.33. The hydropathy profile calculated according to (38) indicates mostly hydrophilic regions, suggesting that cNAPL is a soluble protein. The neural-network based predictors for subcellular localization of proteins ChloroP (31) and LOCtree (32) revealed a putative transit sequence for import into the chloroplast at the N-terminal end of cNAPL, whereas TargetP proposes a mitochondrial localization. However, for chloroplast transit peptides of Chlamydomonas, it was shown that they are more similar to mitochondrial targeting peptides than to chloroplast transit peptides of higher plants (39). The N-terminal amino acid sequence of cNAPL possesses features typical of chloroplast transit peptides, such as an alanine as the second residue. The N-terminal end is an uncharged region with an abundance of arginine (13.6%), valine (13.6%) and alanine (18.2%) residues. This transit peptide could be 46 amino acids long since a possible cleavage site sequence Val-Gly-Ala is present (39). The cleavage would give rise to a predicted mature protein of 304 amino acids with a molecular mass of 34.6 kDa.
Comparison of the genomic and cDNA sequences of cNapl using the C.reinhardtii JGI database (DOE Joint Genome Institute) reveals the presence of 9 large introns and 10 exons (Figure 4). As mentioned above, database searches detected significant sequence similarity with NAPs. Using the C.reinhardtii JGI database, we could also identify a Chlamydomonas homologue of a NAP that does not possess a chloroplast targeting signal. Furthermore, another algal NAP homologue was detected in the marine centric diatom T.pseudonana using the T.pseudonana JGI database (DOE Joint Genome Institute). Figure 5 shows a multiple alignment of cNAPL with NAP polypeptides of algae and higher plants that displayed the highest homology using the BLASTP algorithm (40). Regions of sequence homology between these proteins were found over the entire length of the amino acid sequence, including several blocks of sequence identity. A total of 50 amino acids are identically positioned within all 8 sequences. Overall, there is 29% identity between cNAPL and the other NAP proteins, indicating that cNAPL is a member of the NAP family. With 46% identical amino acids, the Chlamydomonas NAP polypeptide shows a higher degree of homology to plant NAPs than cNAPL. However, Chlamydomonas cNAPL and Thalassiosira NAP share common features atypical for NAPs. They possess, for example, an N-terminal extension that is not found in plant NAPs. In cNAPL, this extension was shown in silico to correspond to a chloroplast targeting signal, whereas an unambiguously prediction is currently impossible for the extension in the sequence of Thalassiosira NAP, because of its complex plastids and multipartite plastid targeting signals (41). Interestingly, no chloroplast transit signal could be identified in all currently available NAP sequences of higher plants.
Figure 4 Physical map of the cNapl gene. Exons (1–10) and introns are represented by closed and open boxes, respectively, with their corresponding size in base pairs. The arrow indicates transcriptional direction. Oligoucleotides used for RT–PCR analysis are given as arrowheads.
When we compared the protein sequences of NAPs from different sources, several evolutionarily conserved domains, such as stretches of acidic residues, as well as nuclear transport signal sequences, were detected (12,42). Recently, Dong and co-worker (42) identified a 16 amino acid sequence in the N-terminal regions of NAP-like sequences from rice and tobacco that is similar to the nuclear export signal (NES) of the yeast NAP1 protein. From the comparison in Figure 5, it is evident that this 16 amino acid stretch is absent in the C.reinhardtii cNAPL sequence. However, there are stretches of amino acid residues in the central and C-terminal part of the sequences of Arabidopsis, maize, pea, rice, soybean and Chlamydomonas NAP that have characteristics of nuclear localization signals (43). The NLS sequences are similar to PKKKKRKV, the NLS found in SV40 T antigen (44). In contrast, cNAPL and Thalassiosira NAP do not possess any NLS sequences. The alignment in Figure 5 reveals two clusters of highly acidic amino acids at the C-terminus of higher plants and Chlamydomonas NAP; both these clusters can also be found in similar positions in the amino acid sequences of yeast NAP1 and HeLa hNRP. Interestingly, however, neither of these clusters is present in cNAPL and Thalassiosira NAP.
Figure 5 Amino acid sequence comparison of C.reinhardtii cNAPL, NAP and NAPs of higher plants and T.pseudonana. Black shading denotes distinct similarities of the tscA-binding protein cNAPL to NAPs. Grey shading shows the higher degree of homology of the NAP protein from C.reinhardtii to NAP proteins of higher plants in contrast to cNAPL. Arrows delimit the NAP domain. A putative prenylation motif is underlined, putative nuclear localization signals are framed and a putative NES is underlined with ‘’. A putative chloroplast targeting signal sequence is represented by ‘+’ on top of the alignment. The two acidic amino acid regions are underlined with dashed lines. An asterisk indicates identical residues. Similar residues are indicated by one or two dots, depending on the degree of similarity of the relevant amino acids. The following protein sequences were used for comparison: At, A.thaliana AAL47354; Cr, C.reinhardtii C_1660026 (JGI); Gm, Glycine max AAA88792; Os, Oryza sativa XP_475795; Ps, Pisum sativum T06807; Tp, T.pseudonana 115707 (JGI); Zm, Zea mays AY100480.
Furthermore, Arabidopsis, maize, pea, rice, soybean and Chlamydomonas NAP contain the CKQQ sequence at their C-terminal ends, a motif suggested as a potential prenylation signal. This signal is supposed to promote membrane interactions and appears to play a major role in several protein–protein interactions. The CKQQ sequence was also identified in NAP polypeptides of rice and tobacco (45). The prenylation signal is missing in cNAPL and Thalassiosira NAP.
To analyse the expression of cNapl in the wild-type strain, RT–PCR analysis was carried out with specific primers designed to amplify cNapl from the start (ATG) to the stop (TAG) codon (Figure 4). As shown in Figure 6a, a single band of around the expected size of 1 kb was obtained. This result indicates the specific expression of cNapl in C.reinhardtii wild-type strain. To examine cNapl expression, total RNA was prepared from wild-type strains CC406 cw15 and arg-cw15 and photosystem I mutants T11-1 59 and T11-3 39 (S. Glanz, unpublished data). Northern blot analysis was carried out using the 32P-labelled 1.7 kb fragment of the cNapl cDNA as probe. As shown in Figure 6b, the probe detected a specific transcript of 1.6 kb, which is consistent with the expected size of the cNapl transcript. An equal load check using Rps18 (46) revealed no difference between transcript accumulation in wild-type versus photosystem I mutant strains. Thus, pleiotropic phenotypes that are often associated with photosynthetic defects do not affect transcriptional expression of the cNapl gene.
Figure 6 Analyses of cNapl transcript. (a) RT–PCR analysis of cNapl transcripts in wild-type arg-cw15. RT–PCR was carried out by using either DNase treated poly(A) RNA as a control (cw15 RNA), cDNA (cw15 cDNA) or genomic DNA (cw15 DNA) as template. Oligonucleotides used for RT–PCR are for_6-19_BspHI and rev_6-19_2xMet_HindIII. Their position is shown in Figure 4. Oligonucleotides rpsF2 and rpsR2 specifically amplify a fragment of the Rps18 transcript and serve as a control for cDNA preparation. Abbreviation: Rps18, cytoplasmic ribosomal protein S18 of C.reinhardtii (46). (b) RNA blot analysis of cNapl transcripts in wild-type and non-photosynthetic mutant strains. Total RNA (20 μg) of wild-type strains CC406 cw15 and arg-cw15 and photosystem I mutants T11-3 39 and T11-1 59 (S. Glanz, unpublished data) were separated on a 1% agarose/formaldehyde gel, transferred onto a nylon membrane and hybridised separately with a cNapl- and Rps18-specific probe as a loading control.
Cellular localization of the cNAPL
The N-terminal 46 amino acids of cNAPL were predicted to be a signal sequence because of certain structural features. To determine the cellular localization of the cNAPL polypeptide, we performed LSCFM. A map of the constructs is shown in Figure 7a. As can be seen in Figure 7b, non-transformed arg-cw15 cells (wt) do not show any fluorescence when green fluorescent protein (GFP) was excited. The complete N-terminal signal sequence (46 amino acids) and the sequence for the first 38 amino acids of the mature cNAPL were fused to a synthetic GFP sequence (47) (N84-cGFP) in frame, keeping the putative N-terminal transit peptide functional. Previous work on diatom protein localization established that the complete pre-sequence of plastid preproteins is sufficient to drive the import of GFP into plastids (41). A similar strategy was successfully applied recently for localization of the phage-type organellar RNA polymerases of Arabidopsis thaliana and Chenopodium album (48). To determine the cellular localization of cNAPL in the algal cell, the cNaplN84-cgfp fusion gene was expressed in C.reinhardtii cells under the control of the HSP70A/RBCS2 promoter (49). As shown in Figure 7b, LSCFM confirmed a plastidic localization of cNAPL. The fluorescent images reveal a co-localization of the chimeric N84-cGFP polypeptide (green fluorescence) with the chloroplast (red autofluorescence of plastids). Right panels show the merged images of cGFP fluorescence and chlorophyll autofluorescence of the same cell. To rule out the possibility that use of the non-native promoter HSP70A/RBCS2 leads to mis-targeting of cNAPL, a gene for a fusion protein with a 31 amino acid deletion in the chloroplast targeting sequence of cNAPLN84 was constructed (N848-39-cGFP). As shown in Figure 7b, this protein clearly localizes in the cytoplasm and to spot-like structures outside of the autofluorescing chloroplast. As further control for nuclear localization, we used cGFP in fusion with the Ble polypeptide (Ble-cGFP). The bacterial ble gene has been successfully expressed in C.reinhardtii by fusing the coding sequence to the 5' and 3' non-coding sequence of the RBCS2 gene, containing additional intron sequences (50). For the Ble polypeptide, it has been shown previously that the bacterial protein is localized in the nucleus (51). This localization was also confirmed by our experiments as demonstrated in Figure 7b and there was no congruence of cGFP fluorescence with the position of the plastid as observed by fluorescence microscopy. In addition, we used the C-terminal cGFP-fused Rps18 polypeptide (Rps18-cGFP) as a marker for cytoplasmic localization. The corresponding cGFP fluorescence signals appeared to accumulate in spots within the cytoplasm. To the best of our knowledge, this is the first demonstration of a cytoplasmic localization of a C.reinhardtii protein using GFP. In conclusion, our results from microscopic investigations clearly demonstrate that the N-terminal extension of cNAPL functions as a chloroplast targeting signal that is able to direct cNAPL to the organelle.
Figure 7 Confocal fluorescence microscopy to demonstrate chloroplast localization of the cNAPL polypeptide. (a) Schematic representation of fusion protein constructs used for cGFP-assays. (b) Laser scanning confocal microscopy of C.reinhardtii arg-cw15 transformants. Transformants were assayed by differential interference contrast microscopy (DIC) or by confocal fluorescence microscopy. DIC, cGFP fluorescence (green) and chlorophyll autofluorescence (red) were merged as indicated. The DIC and cGFP images are representatives of five independent experiments. Scale bar represents 5 μm. Abbreviations: ble, phleomycin resistance gene of Streptoalloteichus hindustanus (50); cgfp, synthetic GFP (47); N84, the N-terminal 84 amino acids of cNAPL; N848-39, the N-terminal 84 amino acids of cNAPL lacking 31 amino acids in the chloroplast targeting sequence; P, HSP70A/RBCS2 promoter (49); Rps18, cytoplasmic ribosomal protein S18 of C.reinhardtii; T, 3'-UTR of Lhcb1 or of RBCS2 gene.
Phylogenetic analyses
The members of the NAP family are considered as histone chaperones, which are conserved from yeast to man. To examine the evolutionary relationships between cNAPL and the above-mentioned homologues, we performed a phylogenetic analysis using the neighbour-joining method. A tree was constructed from multiple alignments of the NAP domains of cNAPL and of NAP polypeptides from various eukaryotes. In addition to the NAP homologue in the diatom T.pseudonana, a partial sequence of a predicted protein with homology to NAPs was found in the EST database of the red alga Porphyra yezoenis (Kazusa DNA Research Institute) (25,26). Similarly, we detected another partial EST homologue (GenBank) from the green alga Dunaliella salina. So far, genomic sequences of these algae are not available yet. For prokaryotes, however, we were unable to detect any NAP or cNAPL homologues in the publicly available genome sequences of bacteria (GenBank) and cyanobacteria (CyanoBase).
The unrooted tree in Figure 8 shows four main clusters: animals, algae, fungi and higher plants. Trees generated with maximum parsimony and Bayesian analysis (52) had an overall similar topography (data not shown). From Figure 8, it becomes evident that the Chlamydomonas NAP polypeptide is related to the group of higher plant NAP proteins, which all lack a chloroplast targeting sequence, whereas cNAPL clusters together with the algal NAP homologues. For the NAP domain, the amino acid sequence of Chlamydomonas NAP shows 54% identity to higher plant NAPs, whereas the NAP domain of cNAPL exhibits 35% identity to higher plant NAPs. The corresponding values for the algal NAPs are 35% for Thalassiosira NAP, 26% for Dunaliella NAP and 15% for Porphyra NAP. Overall, we propose that cNAPL with its chloroplast transit peptide represents a new type of NAP-like polypeptide in photoautotrophic organisms.
Figure 8 Phylogenetic tree of 31 NAP/NAP-like proteins. The tree was generated using PHYLIP (29), as indicated in Material and Methods. Numbers at branches indicate bootstrap support (2000 bootstrap replicates) in percent. Abbreviations: A.f., Aspergillus fumigatus XP_754077; A.t.1, A.thaliana AAL47354; A.t.2, A.thaliana BAA97025; A.t.3, A.thaliana NP_194341; C.n., Cryptococus neoformans AAW43807; D.d., Dictyostelium discoideum EAL71995; D.m., Drosophila melanogaster AAB07898; D.s., D.salina BM448458; G.m., G.max AAA88792; H.s.1, Homo sapiens BAA08904; H.s.2, H.sapiens NP_004528; M.g., Magnaporthe grisea AAX076; N.c., Neurospora crassa CAD70974; N.t.1, Nicotiana tabacum CAD27460; N.t.2, N.tabacum CAD27461; N.t.3, N.tabacum CAD27462; O.s.1, O.sativa AAV88624; O.s.2, O.sativa CAD27459; O.s.3, O.sativa P0456F08; O.s.4, O.sativa XP_475795; P.c., Paramecium caudatum BAB79235; P.s., P.sativum T06807; P.y., P.yezoensis AU191247; R.n.1, Rattus norvegicus AAC67388; R.n.2, R.norvegicus NP_446013; S.c., S.cerevisiae P25293; S.p., Schizzosaccharomyces pombe T41330; T.b., Trypanosoma brucei XP_826968; T.p., T.pseudonana 115707 (JGI).
DISCUSSION
cNAPL is an tscA-specific RNA-binding protein
The splicing chemistry and RNA structures of self-splicing group II introns and nuclear pre-mRNA introns are strikingly similar. Thereby, suggesting some evolutionary relationship between group II introns and the nuclear spliceosomal intron (53). Moreover, there is compelling evidence to suggest that chloroplast spliceosomes catalyse intron splicing using a similar mechanism to that reported for nuclear spliceosomes. This suggestion is the result of data obtained form a combination of different experimental approaches that have identified components of a putative chloroplast spliceosome in C.reinhardtii (7–11) and higher plants (54). Additional support comes from studies with tobacco chloroplasts. Nakamura and co-workers (55) identified chloroplast ribonucleoproteins (cpRNPs) that are associated in vivo with various species of chloroplast mRNAs and intron-containing precursor tRNAs. They suggested that these stromal RNA–protein complexes promote, for example, RNA splicing, processing or editing. Previously, the yeast three-hybrid system was used to demonstrate specific binding of proteins to organellar intron RNA (8,56). In this work, we used the yeast three-hybrid system successfully to screen a C.reinhardtii cDNA library and we were able to detect the novel organellar protein cNAPL that specifically binds to the first psaA group II intron. The binding of cNAPL to domains DII+III and DV+VI of the first psaA intron was further shown in electromobility shift assays.
In several other studies using defined intron domains, separated molecules were found to fold into a structure corresponding to their conformation in the complete active intronic RNA (57,58). Recent studies have demonstrated that fragmented intron molecules are appropriate targets for gel retardation assays (22). In EMSA, we observed multiple shifted bands after incubating domains DII+III of tscA RNA with increasing amounts of cNAPL. As was shown for example for the mutated SxlN1 protein of Drosophila, multiple shifted bands probably correspond to the sequential filling of binding sites when protein concentration is increased (59). Hence, the two shifted bands observed in Figure 2b–d shows non-cooperative binding of cNAPL to domains DII and DIII, thus indicating at least two binding sites. However, we were not able to detect a known RNA-binding motif in cNAPL, such as the RNP motif, the K homology motif (KH) or the RGG (Arg-Gly-Gly) box. Further binding studies with truncated cNAPL constructs could help to determine the exact RNA-binding domain.
cNAPL is phylogenetically related to nuclear localized NAPs and most probably has not been acquired from a cyanobacterial endosymbiont
cNAPL is a member of the multifunctional family of NAPs that are involved in processes which normally take place in the nucleus or the cytoplasm of various eukaryotes (12,42,61). Besides their distinct functions in mitotic events and cytokinesis, NAPs act as histone chaperones, binding to all core histones with a preference for H2A and H2B and working as a deposition factor by transferring NAP1-bound histones to double-stranded DNA. In addition to its nucleosome assembly and histone-binding activity, NAP1 as well as plant NAP1-like proteins may be involved in regulating gene expression and therefore cellular differentiation (45,62,63). NAP1 has also been shown to facilitate transcription factor binding by disruption of the histone octamer through the binding of H2A and H2B (13,64). NAP1 also shuttles histones H2A and H2B as well as a complex of H2A, H2B, H3 and H4 between the template DNA and nascent RNA during transcription (65). To date, however, it is still unknown whether NAP1 interacts directly also with RNA.
Phylogenetic analysis indicates that cNAPL is related to nuclear localized NAP polypeptides. The clustering of cNAPL with algal NAPs and not with NAPs of higher plants could be explained by its chloroplast targeting signal. In the case of Thalassiosira NAP, TargetP predicted a 69 amino acid signal sequence, which could not be assigned clearly to any organelle. Diatoms, such as T.pseudonana, possess so-called ‘complex plastids’ delineated by four distinct membranes. For a nuclear-encoded plastid polypeptide to be directed to the plastids, multiple targeting signals are necessary. Thus, determining a protein's localization in diatoms using today's prediction programs is difficult.
It is now generally accepted that double-membrane-bound chloroplasts are the result of an endosymbiotic event involving a cyanobacterial-like organism early on in evolution. During evolution, the cyanobacterial endosymbiont has lost its autonomy, and this has been accompanied by significant changes of the chloroplast proteome (14,66). A key factor in this process of adoption was the loss of genetic material resulting from gene transfer to the cell nucleus. The vast majority of proteins in present day chloroplasts are encoded by the host nucleus and require N-terminal presequences that target them back to the chloroplast. Therefore, in order to establish a functional organelle–nucleus interaction, chloroplasts have had to import a set of new proteins to adapt their metabolism to the new conditions. Interestingly, a proportion of these proteins do not seem to have been acquired from cyanobacteria (14). Phylogenetic analysis and database research revealed that no NAP homologues could be identified in prokaryotic organisms. We therefore suggest that cNAPL does not originate from an endosymbiotic gene transfer event and that the chloroplast of C.reinhardtii has gained cNAPL as a novel nuclear-encoded factor. The phylogenetic analysis suggests that cNapl originate from a nuclear Nap gene, and is probably the result of a gene duplication event that occurred after separation of higher plants from green algae. We thus propose that cNAPL with its chloroplast transit peptide represents a new type of NAP-like polypeptide.
Is cNAPL a multifunctional protein?
Many group II introns of plants have lost their self-splicing activity during evolution and thus, recruited host-encoded proteins as splicing factors (1,67). The participation of nuclear-encoded proteins in chloroplast splicing could provide a means to control the biogenesis of the photosynthetic apparatus. It has been suggested that these recruited host-encoded proteins probably had other functions in the cell. Many of these proteins show some degree of homology to proteins involved in RNA metabolism. For example, Raa2 of C.reinhardtii shows homology to pseudouridine synthetase; however, such enzymatic activity is not required for trans-splicing of psaA RNA (7). Another example is Mss116p of Saccharomyces cerevisiae, which influences the splicing of all nine group I and all four group II introns in mitochondria (68). Mss116p is related to DEAD-box proteins with RNA chaperone function and may facilitate splicing by resolving misfolded introns. However, it is generally discussed that proteins involved in splicing and interacting with intron RNAs seem to support RNA folding or to stabilize the active conformation, whereas the catalytic potential is clearly located in the RNA itself (69). Furthermore, Mss116p is a bifunctional protein that influences, in addition to splicing of group I and group II introns, some RNA end-processing reactions and translation of a subset of mitochondrial mRNAs. Indeed, for several nuclear-encoded host proteins, it is known that they have additional cellular functions and seem to have been recruited for splicing during evolution (1). Therefore, it has been questioned whether cNAPL is also a multifunctional protein. The analysed interactions of cNAPL with tscA RNA and domains DV and DVI of the first psaA intron implicate a role for cNAPL as a potential tscA RNA processing factor and also as a splicing factor of the first psaA intron. An example for such a bifunctional protein comes from recent work where Rat1 is required for both processing of tscA RNA as well as for splicing of the first psaA intron (8). Another factor involved in tscA RNA processing and splicing of both introns is Raa1-L137H (9), allelic to HN31 (70).
cNAPL binds specifically to domains DII and DVI of the first psaA intron. Domain DII is a phylogenetically less-conserved region and was assigned as functionally unimportant because of its high degree of sequence and structure variations (71). However, Chanfreau and Jacquier (72) could show that the so-called tertiary –' interaction (see also Figure 2a) between DII and DVI of group IIA introns is responsible for a conformational change between the two catalytic transesterification steps. Interestingly, only a few of the known group IIB introns can potentially form this interaction. It is discussed that the majority of the group IIB introns interact with protein factors to change the conformation between the first and the second step (69). The fact that cNAPL interacts with both domains, DII and DVI, implies a role for cNAPL in this conformational change.
Our experiments further demonstrated the binding of cNAPL to domain DV of the first psaA intron. Domain DV is the phylogenetically most conserved sequence and essential for the catalytic splicing reaction. DV shows numerous important tertiary interactions and contains important conserved sequence motifs (69). Overall, we assume that cNAPL presumably has a function in the stabilization and correct folding of the catalytic core of the first psaA intron by interacting with tscA RNA and domains DV and DVI.
In addition, our analyses showed that the binding of cNAPL to tscA RNA was competed efficiently by poly(U) in contrast to other RNA homopolymers. Moreover, cNAPL binds the 5'-UTR of U-rich chloroplast transcripts psbA, psbD, rbcL and rps4 considerably better than the nuclear transcripts Lhcb1 5'-UTR, Lhcb1 3'-UTR, Rps18 and Tba1A. It is proven that the 5'-UTRs of chloroplast transcripts contain the cis-acting determinants for the stabilization of plastid transcripts and are essential for initiation of chloroplast translation by binding trans-acting factors (mostly encoded by nuclear genes) and by interaction with the ribosomal preinitiation complex (73). For example, the stability of the psbD mRNA depends on a nucleus-encoded TPR protein, which is part of a high-molecular weight complex mediating its function via the psbD 5'-UTR (74). For the psbA mRNA it is assumed that a multiprotein complex specifically regulates translation of this mRNA through interaction with a U-rich sequence in the 5'-UTR (75). Furthermore, there are several proteins in Chlamydomonas binding to 5'-UTRs of chloroplast mRNAs, e.g. to 5'-UTRs of atpB, rbcL, rps7 and rps12, that so far have not been characterized and are discussed to be required for translational regulation (76,77). However, the cpRNP complexes seem to be not only involved in regulation of translation, but also in distinct steps of post-transcriptional gene expression, e.g. by facilitating proper RNA folding for intron-containing RNA (78). Recently it was demonstrated that there are two RNP complexes in C.reinhardtii that are involved in trans-splicing of psaA, one in the soluble fraction of the chloroplast and the other one associated with chloroplast membranes, which could provide a link to translation (11).
In conclusion, we have isolated a novel type of group II intron RNA-binding protein using the yeast three-hybrid system. Phylogenetic analysis indicates that the chloroplast located cNAPL protein is encoded by a nuclear gene that probably has evolved from duplication of an ancestral Nap gene. Moreover, it is proposed that cpRNP complexes are global mediators of chloroplast RNA metabolism, which connect transcription and translation in the chloroplast (78). Because of cNAPL's binding to U-rich sequences of chloroplast 5'-UTRs, a more general role for this RNA-binding protein is conceivable, e.g. in translation initiation of several chloroplast transcripts. Thus, cNAPL might represent a component of the cpRNP complex.
ACKNOWLEDGEMENTS
We wish to express our thanks to Profs. Peter Hegemann (Berlin), Christoph Beck (Freiburg) and Dr Ulrich Putz (Hamburg) for the gift of plasmids and Drs Birgit Hoff and Stephan Pollmann for advice in LSCFM and Patricia Cebula and Franziska Thorwirth for help with some experiments. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB480 B3). Funding to pay the Open Access publication charges for this article was provided by DFG (Bonn, Bad Godesberg, Germany).
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