Identification of archaeal proteins that affect the exosome function in vitro
© Luz et al; licensee BioMed Central Ltd. 2010
Received: 13 November 2009
Accepted: 27 May 2010
Published: 27 May 2010
The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors.
Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA.
Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.
The exosome is a 3'-5' exonucleolytic multisubunit complex found in archaea and eukaryotes [1–6]. In yeast, where it was first described, the cytoplasmic exosome comprises ten subunits, whereas the nuclear exosome is formed by eleven subunits . Eight subunits show sequence similarity to exoribonucleases, and for some of them catalytic activity was demonstrated in vitro [1, 8]. Recent data indicate, however, that only the subunits Rrp6p and Rrp44p have intrinsic catalytic activity . In yeast, depletion of individual exosome subunits leads to similar phenotypes [1, 10–12], indicating that only the intact complex is functional in vivo.
Analyses of intra-complex interactions have provided the initial information on the yeast exosome architecture [12–15]. A working model for its structure and composition has been proposed based on mass spectrometry analyses of complexes purified under different conditions and on yeast two-hybrid interaction analyses [16, 17]. According to this model, the RNA binding subunits (Rrp4p, Rrp40p and Csl4p) bind to one side of the RNase PH ring, which is formed by Rrp41p-Rrp45p-Rrp46p-Rrp43p-Mtr3p-Rrp42p , whereas the hydrolytic RNases (Rrp44p and Rrp6p) bind to the opposite side of the ring, although Rrp44p, the largest exosome subunit, may also interact with Rrp4p [16, 17]. The crystal structure of the core human exosome has been recently determined, confirming the predictions for the structure of the eukaryotic complex .
The archaeal exosome is composed of four different subunits, two RNA binding subunits, aCsl4 and aRrp4, and two subunits containing RNase PH domains, aRrp41 and aRrp42 [4, 6]. Three copies of an aRrp41-aRrp42 heterodimer form the RNase PH ring [18–22], which associates with three molecules of aRrp4 and/or aCsl4 [18, 21]. The RNase PH ring has three active sites, responsible for the phosphorolytic RNase activity of the complex, formed in the interface between aRrp41 and aRrp42 [18–22]. aCsl4 and aRrp4 may have a flexible structure, which allows them to interact with RNA and bring it to the central pore of the RNase PH ring  and, subsequently, to the active site. Despite having three active sites, the narrow entry pore of the exosome allows the passage of only one single-stranded RNA molecule [22, 23].
In eukaryotes, the exosome associates with specific protein cofactors in both cellular compartments. In the nucleus, the exosome has been shown to interact with Rrp47p, Mtr4p, the TRAMP complex, and Nop53p, and in the cytoplasm, with the Ski complex [[24–28], reviewed in ]. Rrp47p has been proposed to be a nuclear exosome cofactor required for the processing of stable RNAs . Nop53p is a nucleolar protein that activates the exosome during processing of pre-rRNA 7S to mature 5.8S rRNA . Mtr4p is an RNA helicase and is a subunit of the TRAMP complex, which is involved in RNA polyadenylation, directing the RNA for degradation by the exosome [29–31]. The Ski complex also contains a subunit with helicase activity, Ski2p, and is associated with the exosome in mRNA degradation [32, 33]. The different protein cofactors that associate with the exosome may regulate its function.
Although the archaeal exosome has been shown to associate with other proteins , little is known about archaeal exosome cofactors or regulatory proteins. There are, however, some plausible candidates for exosome regulatory factors. Among them, PaSBDS, a protein encoded by a gene found in the Pyrococcus abyssi exosome operon, of which eukaryotic orthologues (Sdo1p in yeast, SBDS in human and in Trypanosoma) have been shown to be involved in ribosome maturation [4, 35–38]. PaNip7 has been shown to bind RNA in vitro  and its yeast orthologue, Nip7p, is involved in rRNA processing and interacts with the yeast exosome subunit Rrp43p , corroborating the hypothesis of PaNip7 being a Pa-exosome cofactor. Pa1135 is a protein of unknown function, encoded by a gene found in the same operon as the RNase P subunit Rrp30 , and may also be involved in RNA processing. In this work, we show that PaSBDS and PaNip7 affect the exosome activity. PaSBDS competes with the exosome for binding to poly-A RNA, thereby slightly inhibiting its degradation by the complex. PaNip7 binds preferentially to U- and AU-rich RNAs, and strongly inhibits the exosome due to its association with both the exosome complex and the substrate RNA. Pa1135 on the other hand, though also binding U- and AU-rich RNAs in vitro, does not inhibit RNA degradation by the exosome. These findings indicate that PaNip7p and PaSBDS may be exosome regulatory factors. This is the first example of archaeal proteins regulating the exosome by inhibition.
Pa-exosome complexes show different affinities for binding to oligo-RNA in vitro
Although the exosome of several archaeal species have already been characterized [18–23, 41], little is known of the protein factors that might interact with the Pa-exosome and regulate its function. In order to obtain insights into the association of the archaeal exosome with other proteins that are expected to function in RNA processing, a series of experiments were performed with three candidate proteins, PaSBDS, PaNip7, and Pa1135. PaSBDS does not bind poly-rC, binds poly-rU with low affinity, binds 10 nucleotides long poly-rA, but more efficiently longer poly-rA, and binds poly-rAU RNAs (Additional file 1 Figure S1A). PaNip7, has already been shown to bind RNA in vitro with higher affinity for U-rich RNAs , and here we show that PaNip7 also binds a poly-AU RNA (Additional file 1 Figure S1B) that contains complementary sequence and can form both intra- and inter-strand base-pairs. Similar to PaNip7 and PaSBDS, Pa1135 binds poly-AU RNA very efficiently, but differently from PaNip7 and PaSBDS, it does not bind to any of the RNA homopolymers tested (Additional file 1 Figure S1C).
Effects of P. abyssi proteins on the exosome-RNA interaction
Consistent with the results described above, parallel EMSA with Pa1135, PaNip7 and PaSBDS and a poly-rA substrate showed that a band shift can be detected only for PaSBDS (Fig. 1A, lanes 2, 3, and 26, respectively). Incubation of the exosome complexes with RNA in the presence of these proteins revealed their effect on the exosome RNA binding activity. As shown here, although Pa1135 does not bind poly-rA, it inhibits 1.6-fold poly-rA binding by RNase PH ring and 1.7-fold binding by PaCsl4-exosome (Fig. 1B, D). However, Pa1135 does not show a pronounced effect on the PaRrp4-exosome binding to poly-rA (Fig. 1A, lanes 13-15; Fig. 1C). Since the exosome has a much higher affinity for binding to poly-rA than PaNip7, in a direct competition assay the exosome was expected to prevail over PaNip7 for poly-rA binding. Instead, the results show a strong decrease in the intensity of the bands shifted by the exosome in the presence of PaNip7 (Fig. 1A, lanes 16-24). PaNip7 caused a three-fold decrease in poly-rA binding by the RNase PH ring, a 40-fold decrease in binding by the PaRrp4-exosome, and a 20-fold decrease in binding by the PaCsl4-exosome. This result indicates that the decrease in the exosome binding to poly-rA caused by PaNip7 may be due to a direct interaction between PaNip7 and the exosome and not to direct competition for binding to poly-rA.
PaSBDS, on the other hand, binds poly-rA, and was expected to compete with the exosome for binding to this RNA. When PaSBDS and the exosome complexes are incubated with poly-rA, the band corresponding to the free RNA decreases in intensity and bands corresponding to PaSBDS-RNA and exosome-RNA become visible (Fig 1A, lanes 27-35). Poly-rA binding by PaRrp4-exosome was little affected by the presence of PaSBDS, indicating that PaRrp4-exosome has higher affinity for poly-rA than PaSBDS (Fig. 1A, lanes 30-32; Fig. 1C). The observation that PaSBDS-RNA complex results in a smear, instead of a well defined band, also indicates a low stability complex. Surprisingly, however, RNase PH ring and PaCsl4-exosome bound poly-rA more efficiently upon addition of PaSBDS (Fig. 1A, lanes 27-29 and 33-35, respectively). In summary, PaSBDS binds poly-rA and increases RNA binding by the RNase PH ring and the PaCsl4-exosome, but does not strongly affect the PaRrp4-exosome. Pa1135 and PaNip7, do not bind poly-rA with high affinity, but affect the exosome. Pa1135 affects RNA binding by the exosome complexes with lower RNA affinity, RNase PH ring and PaCsl4-exosome, while not affecting the PaRrp4-exosome, which has higher affinity for RNA. PaNip7 inhibits all the exosome complexes, and more strongly PaRrp4-exosome and PaCsl4-exosome.
PaNip7 and PaSBDS interfere with exosome RNA degradation
In the absence of PaNip7, upon addition of increasing amounts of the RNase PH ring, the free RNA band decreases in intensity, while the bands of the RNA-protein complexes, and of the degradation product increase (Fig. 3A, lanes 2-4). When incubated with RNA in the presence of PaNip7, the RNase PH ring seems to degrade only the free RNA, and not the molecules bound to PaNip7, since the free RNA band decreases in intensity while the band corresponding to the degradation product increases, and the PaNip7-RNA band remains mostly unchanged (Fig. 3A, lanes 13-15). In the cases of PaRrp4- and PaCsl4-exosome, due to the presence of the RNA binding subunits, the holo-exosome complexes bind the RNA more efficiently than the RNase PH ring (Fig. 3A, lanes 2-10). Upon incubation of PaRrp4- and PaCsl4-exosome with poly-rAU bound to PaNip7, the free RNA band decreases in intensity while exosome-RNA complexes are detected and the band of degradation products becomes visible (Fig. 3A, lanes 16-21). It is interesting to note that in all cases, much less RNA is degraded in the presence of PaNip7 (Fig. 3A, compare lanes 2-10 to lanes 13-21; Fig. 3B-D).
Although the RNase PH ring is a hexameric complex, whereas PaNip7 is a monomeric protein, the bands formed in the presence of RNase PH ring and PaNip7 run as complexes of approximately the same size. One hypothesis to explain this observation is that it is possible that more than one molecule of PaNip7 bind the same RNA, thereby forming larger RNP complexes. We ruled out the possibility that some of the PaRrp41 and PaRrp42 molecules might not be associated in the form of the RNase PH ring and bound RNA in their monomeric forms because the PaRrp41-PaRrp42 complex was purified by using size exclusion chromatography, selecting for complexes corresponding to the RNase PH ring size (Additional file 2 Figure S2). Furthermore, this complex is active for RNA degradation, confirming that the RNase PH ring was reconstituted in vitro.
Pa-exosome inhibition by PaNip7 depends on its ability to bind RNA
PaNip7 affects the exosome RNA polymerase activity
Direct interaction between PaNip7 and Pa-exosome
The archaeal exosome has been shown to bind, degrade and polyadenylate RNA in vitro [18–22]. Recently, the S. solfataricus exosome activities were analyzed in vitro with respect to the different subunit composition, Mg2+ concentration, and the efficiency of polymerization and degradation of RNA, which depend on the concentration of free phosphate or nucleotide diphosphates . We have previously compared the RNA binding abilities between the Pyrococcus horikoshii apo-exosome RNase PH ring and the P. horikoshii-abysii Rrp4-exosome complex . Here we extended the comparison of the RNA binding abilities, and RNA degradation and polymerization activities of three different P. abyssi exosome complexes, RNase PH ring, PaRrp4-exosome and PaCsl4-exosome. Furthermore, since very little is known of the possible archaeal exosome regulatory factors, here we analyzed the effects of three proteins on the exosome activities when degrading different substrates.
The nine-subunit PaRrp4-exosome binds RNA with higher affinity but degrades RNA less efficiently than PaCsl4-exosome. These different characteristics could be explained by PaRrp4 having higher affinity for RNA than PaCsl4, due to the presence of one S1 and one KH domain in PaRrp4, instead of one S1 and one zinc-ribbon domain in PaCsl4 [4, 18]. Consequently, depending on the RNA binding subunit present in the complex, the structure of the exosome RNA entry pore may undergo conformational changes , leading to the different RNA binding affinities of the exosome complexes and the slower RNA degradation by the PaRrp4-exosome. Although the archaeal exosome binds the substrate RNA directly, it may also interact with various protein factors that are important for directing the complex to the substrate, for opening RNA secondary structures, and possibly for regulating its function.
The bacterial PNPase, which is structurally and functionally related to the exosome, is part of the degradosome, a protein complex involved in rRNA and tRNA processing and in mRNA degradation that is formed by PNPase, RNase E, the helicase RhlB, and enolase [reviewed in ]. In eukaryotes, exosome co-factors include the helicase Mtr4p, the TRAMP complex, Nop53p, Dob1p, Rrp47p, Npl3p, Lsm proteins, the Ski complex, and the Nrd1p/Nab3p complex [24, 26–28, 43]. Yeast Nip7p has been shown to interact with the exosome subunit Rrp43p . Conditional depletion of yeast Nip7p leads to the accumulation of pre-rRNA 27S, a precursor of 5.8S and 25S rRNAs . Nip7p interacts with RNA in vitro and with several proteins known to associate with the pre-rRNA 27S [36, 40, 44]. The yeast and archaeal orthologues of Nip7 contain a PUA domain, which has been previously demonstrated to be involved in RNA interaction [39, 45]. Accordingly, Nip7p and PaNip7 bind RNA in vitro, with higher affinity for poly-U RNAs . Here we show that PaNip7 also has high affinity for poly-AU RNAs that can form weak secondary structures. PaNip7 strongly inhibits the archaeal exosome, and this inhibition depends on PaNip7 ability to bind RNA and to interact with the exosome complex. Supporting this conclusion, PaNip7 mutants that do not bind RNA, have smaller inhibitory effects on the exosome. It is possible that PaNip7 binds RNA through its C-terminal PUA domain, and interacts with the archaeal exosome via its N-terminal domain, thereby controlling the exosome function. The hypothesis that the inhibition of the exosome by PaNip7 involves both RNA binding and protein interaction is further strengthened by the observations that PaNip7 also inhibits the degradation of RNAs for which PaNip7 has low affinity, such as poly-rA. Interestingly, PaNip7 has stronger inhibitory effect on PaCsl4-exosome, indicating a possible role for PaNip7 as a regulatory factor for one of the exosome complexes. PaNip7 could interact with PaCsl4 Zn-ribbon domain, which may be involved in the interaction with proteins, providing a mechanism for the exosome regulation.
Pa1135 was shown here to bind poly-rAU RNA but contrary to PaNip7 does not strongly affect the exosome RNase activity, although Pa1135 inhibits RNA binding by the RNase PH ring and PaCsl4-exosome complexes. The P. horikoshii RNase P is formed by the RNA component and five proteins, including Rrp30 . Since Pa1135 is encoded by a gene found in the same operon as the RNase P subunit Rrp30 , it is possible that Pa1135 regulates RNase P function in vivo.
Although eukaryotic SBDS/Sdo1p has not been shown to interact with the exosome, it has been shown to be required for pre-rRNA processing, for 60S ribosomal subunit translational activation and Tif6p recycling . In addition, SBDS has been shown to interact with the human orthologue of Nip7p and its deficiency affects expression of different genes . Yeast Sdo1p has recently been shown to interact with Nip7p and to bind poly-A and poly-AU RNA . The PaSBDS gene is found in the same operon as three of the exosome subunits, indicating that it is also involved in RNA metabolism.
Structural analysis of Archeoglobus fulgidus SBDS has shown that this protein contains an RNA binding domain . In this work, we confirmed the hypothesis of PaSBDS binding to RNA, by showing that it binds poly-rA, poly-rU and poly-rAU in vitro in a length-dependent manner, and competes with the PaRrp4-exosome for binding to A-rich RNAs. These results indicate that PaSBDS is also involved in RNA processing and may regulate one of the archaeal exosome complexes in vivo. Further indication of exosome regulation was obtained in RNA degradation assays, in which the RNase activity of the RNase PH ring and of the PaRrp4-exosome was slightly inhibited by PaSBDS. It is therefore possible that PaSBDS interacts with RNA in vivo and controls its processing by the exosome.
We show in this work that two archaeal proteins that bind RNA can affect the Pa-exosome activity, making them candidates to be exosome regulatory factors. PaSBDS binds A- and AU-rich RNAs and inhibits mainly the PaRrp4-exosome. PaNip7 binds preferably AU-rich RNAs and strongly inhibits PaCsl4-exosome. Furthermore, similar to the eukaryotic counterparts, PaNip7 interacts with the Pa-exosome. Based on the results shown here that PaSBDS and PaNip7 inhibit preferentially specific exosome complexes, it is possible that these proteins control the Pa-exosome in vivo. The evolutionary conserved structures and RNA affinities of Nip7 and SBDS raise the hypothesis that their eukaryotic orthologues also control the exosome function.
Microorganisms, Plasmids, Enzymes, and DNA Manipulation
The Escherichia coli strains used in this study were DH5α and BL21-CodonPlus (DE3)-RIL (Stratagene). Genomic DNA of P. abyssi GE5 was kindly provided by Dr. Patrick Forterre (Institut de Génétique et Microbiologie, Université Paris Sud, France). Plasmid DNA was extracted using Qiagen plasmid purification systems. Restriction enzymes and other DNA-modifying enzymes were used as recommended by the manufacturer (New England Biolabs).
Construction of Expression Vectors
Plasmids for E. coli expression of PaRrp4 , wild type PaNip7  and mutants PaNip7R151A, R152A and PaNip7K155A, K158A  have been described previously. PaSBDS (PAB0418) was PCR amplified from P. abyssi genomic DNA using primers PaSBDSfor (5'-AGGATCCCATATGCCTATTAGCGTTG-3') and PaSBDSrev (5'-CGGCCTCGAGTCATAGCCCCTT-3'), digested with BamH I-Xho I and inserted into vector pET-28a (Novagen), also digested with BamH I-Xho I. Pa1135 coding sequence was PCR-amplified from P. abyssi genomic DNA using primers Pa1135for (5'-GTTAGGGGGGATCCATGGCAG-3') and Pa1135rev (5'-CGGCCTCGAGTCAATCCTCCC-3'), and inserted into vector pET28a, digested with BamH I-Xho I. The PaRRP41 coding sequence was amplified from P. abyssi genomic DNA using primers PaRRP41for (5'-TACTCGAGCATATGATGGAGAAACCAGAAG-3') and PaRRP41rev (5'-ACGAATTCATCTCATTATCACTCACTTTC-3') and inserted into the PCR product cloning vector pGEM-T (Promega) prior to subcloning into the Nde I and Sal I restriction sites of the expression vector pET29a (Novagen). PaRRP42 was amplified using primers PaRRP42for (5'-AGGGATCCCATATGAGTGATAATGAGATCG-3') and PaRRP42rev (5'-TACGCGTATCGATGTTATATCATTGCTTTGC-3'), inserted into pGEM-T vector (Promega) and subsequently subcloned into the BamH I and EcoR I sites of the expression vector pAE . PaCSL4 was amplified using primers PaCSL4for (5'-AGATCTCATATGGAGGAAGGTGAGGAG-3') and PaCSL4rev (5'-AGAATTCTTTGCCCTCATAGCTTC-3') and inserted into the Nde I and EcoR I sites of the expression vector pET28a (Novagen).
Expression and Purification of Proteins
Recombinant proteins were expressed in the E. coli BL21-CodonPlus (DE3)-RIL strain, transformed with the correspondent plasmids. Expression was induced by addition of 10 mM lactose or, in the case of PaSBDS, 0.5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). Cells expressing PaRrp41 and PaRrp42 were harvested and suspended in buffer A (30 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM imidazole). Cells expressing PaSBDS were suspended in buffer B (30 mM Tris-HCl, pH 7.0, 500 mM NaCl, 5 mM imidazole). Cells expressing Pa1135 were suspended in buffer C (30 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole). All cells were lysed in a French press. The lysate was heated at 85°C for 30 min and cooled on ice for 15 min. After centrifugation at 20,000 × g for 30 min, the supernatant was fractionated by affinity chromatography in Ni-NTA-agarose (Qiagen), followed by gel filtration in superdex 75 (GE Healthcare).
PaNip7, PaNip7R151A, R152A and PaNip7K155A, K158A were purified as described previously [39, 48]. To reconstitute the archaeal exosome, the RNase PH ring complex was obtained by co-expressing the proteins PaRrp41 and PaRrp42 in E. coli, transformed with the plasmids pET29-PaRrp41 (KanR) and pAE-PaRrp42 (AmpR). For PaRrp4-exo assembly, cells expressing PaRrp4 were mixed with cells expressing PaRrp41 and PaRrp42, prior to lysis. PaCsl4 was obtained by co-expression with PaRrp42. PaCsl4+PaRrp42-expressing cells were mixed to PaRrp41+PaRrp42-expressing cells to obtain the PaCsl4-exo. Protein samples were concentrated by centrifugation using Centricon microconcentrators (Millipore). Protein content was determined by bicinchoninic acid (BCA) assay for protein quantitation (Sigma).
RNase PH Phosphorolysis and Polymerase Assays
RNA phosphorolysis was assayed by incubating the complexes with 1 pmol 32P-labeled RNAs in Tris-50 buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 100 μg/ml BSA), and 10 mM NaH2PO4, in 10 μl at 65°C for 15 minutes. RNA polymerization was accomplished under the same conditions, but using ADP instead of NaH2PO4, in 20 μl at 37°C for 30 minutes. RNA degradation and polymerization products were resolved on 8% or 10% denaturing polyacrylamide gel and visualized on a Phosphorimager (MolecularDynamics).
RNA binding assay
RNA binding assays were carried out with 1 pmol 32P 5'-labeled 14-mer poly-rA, poly-rU, 13-mer poly-rC, and 21-mer poly-rAU (5'-UUAUUAUUUAUUUAUUAUUUA-3') oligoribonucleotides (IDT). The assays were performed under the same conditions as described previously  or in 20 mM Tris-HCl pH 8.0, 20 mM KCl, 2 mM MgCl2 1 mM DTT, 100 μg/ml BSA, 0.8 U RNasin. Different concentrations of proteins were incubated with the substrate RNA in 20 μl at 37°C for 30 minutes. The samples were resolved on 8% native polyacrylamide gels and visualized on a Phosphorimager (MolecularDynamics).
RNA protection assays
RNA protection assays were carried out with 1 pmol 32P 5'-labeled poly(rAU) oligoribonucleotides. Protein cofactors were incubated with the RNAs for 30 min at 37°C, after which time the exosome complexes and NaH2PO4 (to the final concentration of 10 mM) were added to the reactions, which were incubated at 65°C for further 15 min. The products were resolved on native or denaturing 8% polyacrylamide gels and visualized on a Phosphorimager (MolecularDynamics).
Quantitative analysis of exosome activities in vitro
A Phosphorimager (MolecularDynamics) was used for quantitation of the bands obtained from RNA degradation, polyadenylation and binding assays.
Protein interaction assays
PaNip7 was labeled with FITC (fluorescein isothiocyanate), by following the instructions of the manufacturer's protocol (Molecular Probes). 12.5 μg of PaNip7-FITC were incubated with 25 μg of the exosome complexes at 25°C for 30 minutes. The samples were subsequently subjected to electrophoresis on native 6% polyacrylamide gels, and the fluorescence was analyzed on a Phosphorimager (MolecularDynamics). Concomitantly, samples were subjected to size exclusion chromatography and the absorbance was monitored at 280 nm and the fluorescence was monitored at 520 nm.
J.S.L., C.R.R.R., M.C.T. and P.P.C. were recipients of FAPESP fellowships. F.L.P. was a recipient of a CNPq fellowship. This work was supported by FAPESP grants (05/56493-9 and 07/57096-9 to C.C.O.).
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