Rute Gonçalves Matos

Ana Barbas

Cecília Maria Arraiano

RNase R mutants elucidate the catalysis of structured RNA: RNA-binding Domains select the RNAs targeted for degradation

Keywords: Ribonuclease, RNase II, RNase R, Exosome, RNA degradation, structured RNA 3D, three-dimensional, CSD, cold shock domain, RNB, RNase II catalytic domain, HTH, helix-turn-helix, IPTG, isopropyl-(3-D-thiogalactoside), DTT, dithiotreitol, PMSF, phenylmethylsulfonyl fluoride, SPR, Surface Plasmon Ressonance

The RNase II super family is an ubiquitous family of exoribonucleases essential for RNA metabolism. RNase II and RNase R degrade RNA in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading highly structured RNAs, the RNase II activity is impaired by the presence of secondary structures. RNase II and RNase R share structural properties and have a similar modular domain organization. The eukaryotic RNase II homologue, Rrp44/Dis3, is the catalytic subunit of the exosome, one of the most important protein complexes involved in the maintenance of the correct levels of cellular RNAs. In this work we constructed truncated RNase II and RNase R proteins and point mutants and characterized them regarding their exoribonucleolytic activity and RNA-binding ability. We report that Asp-280 is crucial for RNase R activity without affecting RNA binding. When Tyr-324 was changed to alanine the final product changed from 2 to 5nts showing that this residue is responsible for setting the end-product. We have shown that the RNB domain of RNase II has catalytic activity. The most striking result is that the RNase R RNB domain per se degrades double stranded substrates even in the absence of a 3'-overhang. Moreover, we demonstrate for the first time that the substrate recognition of RNase R depends on the RNA binding domains that target the degradation of RNAs that are "tagged" by a 3'-tail. These results can have important implications for the study of poly(A)dependent RNA degradation mechanisms.

Introduction

E. coli RNase II is the prototype of the RNase II super family of exoribonucleases. Homologues of RNase II/R are present in all domains of life [START_REF] Grossman | RNase II structure completes group portrait of 3' exoribonucleases[END_REF][START_REF] Mian | Comparative sequence analysis of ribonucleases HII, III, II PH and D[END_REF][START_REF] Mitchell | The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'[END_REF][START_REF] Zuo | Exoribonuclease superfamilies: structural analysis and phylogenetic distribution[END_REF]. RNase R is a member of this family that is involved in mRNA degradation, in RNA and protein quality control and has been shown to be required for virulence [START_REF] Cheng | The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R[END_REF][START_REF] Cheng | An important role for RNase R in mRNA decay[END_REF][START_REF] Cairrão | The role of endoribonucleases in the regulation of RNase R[END_REF][START_REF] Cairrão | Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA[END_REF][START_REF] Andrade | RNase R affects gene expression in stationary phase: regulation of ompA[END_REF]. In the nucleus and cytoplasm of eukaryotic cells, the RNase II homologue Rrp44/Dis3 is a subunit of the exosome, an essential multiprotein complex, involved in the processing, turnover and quality control of different types of RNAs [START_REF] Mitchell | The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'[END_REF]. Rrp44/Dis3 is the only catalytically active nuclease in the yeast core exosome [10] and recently it was shown that it contains both exo and endoribonuclease activity [11,[START_REF] Schaeffer | The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities[END_REF]. RNase II and RNase R share catalytic properties: they both degrade RNA processively, in the 3' to 5' direction releasing 5'-nucleotide monophosphates. These enzymes also share structural properties, including 60% sequence homology [13]. Their activity is sequence independent but while RNase II is sensitive to secondary structures, RNase R is capable of degrading highly structured RNA [START_REF] Cheng | An important role for RNase R in mRNA decay[END_REF][START_REF] Andrade | RNase R affects gene expression in stationary phase: regulation of ompA[END_REF]13,[START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF]. Another difference is that the final degradation product of RNase II is a 4 nucleotide fragment, whereas the end-product of RNase R is a 2 nucleotide fragment [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF][START_REF] Amblar | The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization[END_REF][START_REF] Cannistraro | The processive reaction mechanism of ribonuclease II[END_REF]. RNase R is a 92 KDa protein encoded by the rnr gene. It is involved in the degradation of different types of RNAs such as rRNAs, sRNAs and mRNAs. It was shown that RNase R has in vivo affinity for polyadenylated RNA and that it can be a key enzyme involved in poly(A) metabolism [START_REF] Andrade | The poly(A)dependent degradation pathway of rpsO mRNA is primarily mediated by RNase R[END_REF]. RNase R is the only known 3' to 5' exoribonuclease able to degrade through double stranded RNA without the aid of helicase activity [START_REF] Andrade | The role of 3'-5' exoribonucleases in RNA degradation[END_REF]. It is a cold shock protein that is regulated transcriptionally and post-transcriptionally [START_REF] Cairrão | The role of endoribonucleases in the regulation of RNase R[END_REF][START_REF] Cairrão | Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA[END_REF]. The activity of RNase R is modulated according the growth conditions of the cell [19] and its levels increase in stationary phase and under stress conditions [START_REF] Cheng | An important role for RNase R in mRNA decay[END_REF][START_REF] Cairrão | Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA[END_REF][START_REF] Andrade | RNase R affects gene expression in stationary phase: regulation of ompA[END_REF]. It has been shown that this protein is also involved in pathogenesis in different microorganisms [START_REF] Cheng | The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R[END_REF][START_REF] Erova | Cold shock exoribonuclease R (VacB) is involved in Aeromonas hydrophila pathogenesis[END_REF][START_REF] Tobe | vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri[END_REF][START_REF] Tsao | The 3'-to-5' exoribonuclease (encoded by HP1248) of Helicobacter pylori regulates motility and apoptosis-inducing genes[END_REF]. RNase II is a 72 KDa protein encoded by gene rnb. In E. coli, this protein is the major hydrolytic enzyme that is responsible for 90% of the exoribonucleolytic activity in crude extracts [START_REF] Deutscher | Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis[END_REF]. RNase II expression is differentially regulated at the transcriptional and posttranscriptional levels and the protein can be regulated by the environmental conditions [19, [START_REF] Zilhão | PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism[END_REF][START_REF] Zilhão | DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II[END_REF]. The determination of the three-dimensional structure of E. coli RNase II showed that RNase II consists of 4 domains: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain [26,[START_REF] Zuo | Structural basis for processivity and single-strand specificity of RNase II[END_REF] (Figure 1). The structural and biochemical analysis helped explaining the activity of the enzyme and led to the proposal of a model for RNA degradation by RNase II. RNA contacts RNase II in two different and non-contiguous regions, the anchoring and the catalytic regions [START_REF] Cannistraro | The processive reaction mechanism of ribonuclease II[END_REF]26]. The shortest RNA substrate able to retain contacts with these two regions is a 10-nucleotide fragment and it was known that this was the minimum size necessary to maintain the processivity of the enzyme [START_REF] Cannistraro | The processive reaction mechanism of ribonuclease II[END_REF]26,28]. It was demonstrated that Tyr-313 and Glu390 are important to the discrimination of cleavage of RNA versus DNA [START_REF] Barbas | Determination of key residues for catalysis and RNA-cleavage specificity: one mutation turns RNase II into a "super"-enzyme[END_REF]. During the determination of key residues for catalysis, we have recently discovered that the substitution of the Glu-542 by alanine leads to a 100-fold increase in the exoribonucleolytic activity, turning RNase II into a "super-enzyme" [START_REF] Barbas | Determination of key residues for catalysis and RNA-cleavage specificity: one mutation turns RNase II into a "super"-enzyme[END_REF]. The structure of the RNA bound complex allowed explaining why 4nt is the final degradation product for RNase II [26]. This is a result of the tight packing of the five 3´-terminal nucleotides in the catalytic cavity, mediated by the aromatic residues Tyr-253 and Phe-358 [26]. It was reported that the highly conserved residue Tyr-253 is the one responsible for setting the end-product of RNase II and it is extremely important for the maintenance of the "RNA clamping" in the catalytic site of RNase II [28].

The structural model of E. coli RNase R protein was constructed based on the RNase II structure; the two enzymes share a common 3D arrangement, being all the critical residues for exoribonucleolytic activity located in equivalent spatial positions [28]. By comparing the protein models it was noteworthy that these two proteins have a common arrangement of the clamping tyrosine amino acid but, in contrast, the presence of Phe-358 is exclusive to RNase II. RNase R protein presents a Phe residue in the immediate downstream position (Phe-429), perhaps with a similar functionality in fixing the RNA. The differences in the equivalent amino acid Phe-358 in RNase II and RNase R could explain their differences in regard to RNA degradation [28]. It was also shown that the conserved Asp-209 residue is directly involved in RNA cleavage by RNase II. This is the only critical aspartate for RNase II activity [28,30], and it's substitution by Asn was responsible for the total loss of the protein activity without affecting its RNA binding ability [30]. Moreover, a similar mutation in the yeast RNase II homologue Rrp44/Dis3 (D551N) totally abolished activity without reducing substrate binding. This mutation was also responsible for a very strong growth defect, suggesting that the phenotype of this mutant is very important for yeast physiology [10,[START_REF] Schneider | The exosome subunit Rrp44 plays a direct role in RNA substrate recognition[END_REF]. In this work we constructed and analyzed the equivalent mutants to Tyr253Ala and Asp209Asn of E. coli RNase II in RNase R, Tyr324Ala and Asp280Asn, respectively. Our aim was to confirm their importance in the activity of the enzymes of the RNase II family. We observed that the mutant Asp280Asn has no activity but is still able to bind RNA efficiently, which confirms the importance of this aspartate in catalysis. When Tyr-324 was changed into an alanine, we verified that the final product of RNase R changed from 2 to 5 nts, confirming the importance of this residue in setting the final-end product in this family of enzymes. Furthermore, we constructed a set of truncated proteins of RNase R lacking CSD and/or S1 domains in order to understand the contribution of each domain (Figure 1). We analyzed the activity and the binding ability of the truncated proteins using different substrates and observed that the RNB domain of RNase R alone is capable of degrading RNA molecules. In addition, we show that this domain is sufficient for the degradation of structured substrates, and that is also able to bind to RNA, although with less efficiency when compared with the wild type protein. We also observed that the CSD and S1 domains are responsible for the binding of the proteins to the substrate and that they are crucial for the recognition of which substrates are targeted to be degraded. In the absence of both these domains the protein is capable of degrading perfect double stranded RNAs that lack the 3´end overhang. To date it had always been reported that the existence of a 3´end overhang of at least 5nts length for RNase R was essential for catalysis to occur; here we unravel the reason why.

Experimental

Restriction enzymes, T4 DNA ligase, Pfu DNA polymerase and T4 Polynucleotide Kinase were purchased from Fermentas, Unlabeled oligonucleotide primers were synthesized by STAB Vida, Portugal.

Strains

The E. coli strains used were DH5α (F' fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17a) [START_REF] Taylor | E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing[END_REF] for cloning experiments and BL21(DE3) (F -r B -m B -gal ompT (int::P lacUV5 T7 gen1 imm21 nin5) [START_REF] Studier | Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes[END_REF] for expression and purification of enzymes.

Construction of RNase R mutants by PCR Overlapping

The point mutations Asp280Asnand Tyr324Ala were introduced into pABA-RNR (Table 1) by PCR Overlapping. The primers used in the construction of RNase R mutants were Asp280Asn_Fw, Asp280Asn_Rev, Tyr324Ala_Fw and Tyr324Ala_Rev (Table 2). All mutant constructs were confirmed by DNA sequencing at STAB Vida, Portugal.

Construction of truncated proteins

The truncated proteins were constructed by removing different regions of rnb and rnr genes in pFCT6.1 and pABA-RNR plasmids (Table 1). XhoI restriction site was introduced into pFCT6.1 in the position 512 by overlapping PCR using the primers pFCT_XhoI512_Fw and pFCT_XhoI521_Rev (Table 2) originating the pFCT_XhoI521 plasmid. The natural occurrence of an XhoI recognition site in the position 69 allowed us to construct the pFCT_ΔCSDs by digesting the plasmid pFCT_XhoI521 with XhoI (that originated degradation products with 6955 bps and 456 bps, the last one corresponding to CSD1 and CSD2 of RNase II) and circularizing the fragment with 6955 bps. The protein RNase II_RNB was constructed by introduction of a stop codon (TAA) at the position 1735 in the plasmid pFCT_ΔCSDs using the primers RNB_Stop1735_Fw and RNB_Stop1735_Rev (Table 2). In the pABA-RNR plasmid (Table 1) it was introduced the NdeI restriction site in the position 549 by overlapping PCR using the primers RNR_NdeI549_Fw and RNR_NdeI549_Rev (Table 2) originating the pABA-RNR_NdeI549 plasmid. The natural occurrence of an NdeI recognition site in the position 60 allowed us to construct the pABA-RNR_ΔCSDs by digesting the plasmid pABA-RNR_NdeI549 with NdeI (that originated degradation products with 7624 bps and 651 bps, the last one corresponding to HTH, CSD1 and CSD2 of RNase R) and circularizing the fragment with 7624 bps. The proteins RNR_ΔS1 and pABA-RNR_RNB were constructed by introduction of a stop codon (TAA) at the position 1927 in the plasmids pABA-RNR and pABA-RNR_ΔCSDs, respectively, using the primers RNR_Stop1927_Fw and RNR_Stop1927_Rev (Table 2).

Overexpression and purification of wild type and RNase II mutants

The plasmid used for expression of wild-type E. coli histidine-tagged RNase II protein was pFCT6.1 plasmid [19] (Table 1). This plasmid contains the rnb gene cloned into pET-15b vector (Novagen) under the control of φ10 promoter, allowing the expression of the (His) 6tagged RNase II fusion protein. The plasmid used for expression of wild-type E. coli histidine-tagged RNase R protein was pABA-RNR (Table 1) [START_REF] Amblar | The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization[END_REF] plasmid that contains the rnr gene cloned into pET-15b vector (Novagen) under the control of φ10 promoter, allowing the expression of the (His) 6 -tagged RNase R fusion protein. All plasmids were transformed into BL21(DE3) E. coli strain (Novagen) to allow the expression of the recombinant proteins. Cells were grown at 30ºC in 100 ml LB medium supplemented with 150 µg/ml amplicillin to an A 600 of 1.5. Then, they were transferred to 18ºC for 30 minutes and then induced by addition of 0.5 mM IPTG; induction proceeded for 20 hours at 18ºC. Cell cultures were pelleted by centrifugation and stored at -80°C. Purification of all proteins was performed by histidine affinity chromatography using HiTrap Chelating HP columns (GE Healthcare) and AKTA HLPC system (GE Healthcare) following the protocol previously described [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF][START_REF] Arraiano | Characterizing ribonucleases in vitro examples of synergies between biochemical and structural analysis[END_REF]. Briefly, cell suspensions were lysed using a French Press at 9,000 psi in the presence of 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The crude extracts were treated with Benzonase (Sigma) to degrade the nucleic acids and clarified by a 30 min centrifugation at 10,000 g. The clarified extracts were then added to a HiTrap Chelating Sepharose 1 ml column equilibrated in buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH8) plus 20 mM imidazol and 2 mM β-mercaptoethanol. Protein elution was achieved by a continuous imidazol gradient (from 20 mM to 500 mM) in buffer A. The fractions containing the purified protein were pooled together and buffer exchanged to buffer B (20 mM Tris-HCl pH8, 100 mM KCl and 2 mM β-mercaptoethanol) using a desalting 5 ml column (GE Healthcare). Eluted proteins were concentrated by centrifugation at 15 ºC with Amicon Ultra Centrifugal Filter Devices of 30,000 MWCO (Millipore). Protein concentration was determined by spectrophotometry and 50% (v/v) glycerol was added to the final fractions prior storage at -20 ºC. 0.5 μg of each purified protein was applied in a 8% SDS-PAGE and visualized by Coomassie blue staining (data not shown).

Activity assays

Exoribonucleolytic activity was assayed using three different RNA oligoribonucleotides as substrates [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF][START_REF] Arraiano | Characterizing ribonucleases in vitro examples of synergies between biochemical and structural analysis[END_REF]. The 30mer oligoribonucleotide (5'-CCCGACACCAACCACUAAAAAAAAAAAAAA-3'), the 16mer oligoribonucleotide (5'-CCCGACACCAACCACU-3') and the poly(A) chain of 35nts were labelled at its 5'-end with [γ- 32 ATP] and T4 polynucleotide kinase. The RNA oligomers were then purified using Microcon YM-3 Centrifugal Filter Devices (Millipore) to remove the non-incorporated nucleotides. The labelled 30mer and 16mer oligoribonucleotides were hybridized to the complementary 16mer oligodeoxiribonucleotide (5'AGT GGT TGG TGT CGG G 3'), thus obtaining the corresponding double stranded substrate 16-30ds and 16-16ds respectively. The hybridization was performed in a 1:1 (mol:mol) ratio in the Tris component of the activity by 5 min of incubation at 68ºC followed by 45 min as room temperature. The exoribonucleolytic reactions were carried out in a final volume of 10 μl containing 30 nM of substrate, 20 mM Tris-HCl pH8, 100 mM KCl, 1 mM MgCl 2 , and 1 mM DTT. The amount of each enzyme added to the reaction is indicated in the respective Figures. Reactions were started by the addition of the enzyme and incubated at 37ºC. Samples were withdrawn at the time points indicated in the Figures and the reaction was stopped by adding formamide-containing dye supplemented with 10 mM EDTA. Reaction products were resolved in a 20% polyacrylamide/7 M urea and analyzed by autoradiography. The exoribonucleolytic activity of the enzymes was determined by measuring and quantifying the disappearance of the substrate in several distinct experiments in which the protein concentration was adjusted in order that, under those conditions, less than 25% of substrate was degraded. Each value obtained represents the mean of these independent assays. The exoribonucleolytic activity of the wild-type enzymes was taken as 100%.

Surface plasmon resonance analysis -BIACORE

Biacore SA chips were obtained from Biacore Inc. (GE Healthcare). The Flow cells of the SA streptavidin sensor chip were coated with a low concentration of the following substrates. On flow cell 1 no substrate was added so this cell could be used as the control blank cell. On flow cell 2 a 5' biotinylated 25-nucleotide RNA oligomer (5'-CCC GAC ACC AAC CAC UAA AAA AAA A-3') was added while on flow cell 3 we added a 5' biotinylated 30-mer poly(A) oligomer to allow the study of the protein interaction with different single-stranded RNA substrates. The target substrates were captured on flow cells 2 and 3 by manually injecting 20 μl of a 500 nM solution of the substrates in 1 M NaCl at a 10 μl/min flow rate, as described in previous reports [28,[START_REF] Barbas | Determination of key residues for catalysis and RNA-cleavage specificity: one mutation turns RNase II into a "super"-enzyme[END_REF][START_REF] Arraiano | Characterizing ribonucleases in vitro examples of synergies between biochemical and structural analysis[END_REF]35]. The biosensor assay was run at 4°C in the buffer with 20 mM Tris-HCl pH8, 100 mM KCl, 1 mM DTT and 25 mM EDTA. The proteins were injected over flow cells 1, 2 and 3 for 2 min at concentrations of 10, 20, 30, 40 and 50 nM using a flow rate of 20μl/min. All experiments included triple injections of each protein concentration to determine the reproducibility of the signal and control injections to assess the stability of the RNA surface during the experiment. Bound protein was removed with a 60-s wash with 2 M NaCl, which did not damage the substrate surface. Data from flow cell 1 were used to correct for refractive index changes and non-specific binding. Rate constants and equilibrium constants were calculated using the BIA EVALUATION 3.0 software package, according to the fitting model 1:1 Languimir Binding.

Multiple sequence alignment.

Homologous sequences belonging to the to RNase II family of proteins in protein databases were obtained using Blast [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] and they were aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] and T-COFFEE [START_REF] Notredame | T-Coffee: A novel method for fast and accurate multiple sequence alignment[END_REF] algorithms.

Results and Discussion

Arg-280 is crucial for RNase R activity without affecting RNA binding ability. The active site of RNase II has four conserved aspartic residues that are the responsible for positioning the RNA substrate correctly in order to promote the nucleophilic attack of the phosphodiester bond [26]. Previous studies have shown that, in this protein, Arg-209 is a crucial residue for the catalytic activity without affecting RNA binding [30] and that this arpartate is the only one that is essential for the activity of RNase II [28]. Since this is a highly conserved residue in this family of enzymes (Supplementary Figure 1), we also mutated the correspondent amino acid in RNase R, Arg-280, to better understand the reaction mechanism of this exoribonuclease. For that, we introduced the point mutation Arg280Asn into the pABA-RNR [START_REF] Amblar | The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization[END_REF], and overexpressed and purified the respective protein. The exoribonucleolytic activity of this mutant protein was analyzed and compared to the wild type by performing activity assays using different types of RNA substrates. Wild type RNase R is able to degrade single and double-stranded RNA substrates releasing 2nts fragment as the shortest final end-product in both cases (Figure 2). In fact, the final endproduct is a mixture of a 2nts and 4nts fragments (Figure 2). The structure of RNase II showed that in the catalytic pocket 5nts of RNA were clamped between the aromatic residues Tyr-253 and Phe-358 [26]. Tyr253 is highly conserved and equivalent residues are present in all RNase II family members (Supplementary Figure 1). However in RNase R the equivalent residue to Phe-358 does not exist and that could be a possible explanation for the differences regarding the final end-product [28]. In RNase R there is a Phe residue (Phe-429) in the position immediately downstream of the equivalent residue of Phe-358 in RNase II. It is possible that some RNA fragments are still partially "clamped" and therefore they are released when they reach 4nts. Others bind more efficiently the catalytic cavity and are able to be degraded up to a 2nts fragment. This issue will be clarified when the RNase R crystal structure is solved. When the D280N mutant of RNase R was tested with both single and double stranded substrates, no activity was found (Figure 2), similarly to what happened with RNase II_D209N mutant [28,30]. Furthermore, we determined the exoribonucleolytic activity of wild-type RNase R and D280N mutant enzymes. Our results are consistent and confirm those obtained with the activity assays, being the activity of D280N mutant highly reduced when compared to the wild type enzyme (Table 3). To determine the involvement of this residue in RNA binding we calculated the dissociation constants of the wild type and mutant proteins by Surface Plasmon Resonance (SPR) using two different RNA substrates, a 25-mer and a poly(A) oligomers. We observed that the binding ability of the protein is not affected by the presence of this mutation, since D280N mutant presented a K D value very similar to the wild type RNase R protein with both substrates tested: 2.2 ± 0.3 nM with 25mer and 1.3 ± 0.2 nM with poly(A) for D280N versus 3.2 ± 0.4 nM with 25-mer and 1.2 ± 0.1 nM with poly(A) for RNase R (Table 4). These results confirm that, similarly to what happens with RNase II, this aspartate is indeed essential for the activity of RNase R without affecting RNA binding. In RNase R, the four aspartates in the active center are located in the positions 272, 278, 280 and 281 and, like Asp-209 in RNase II, the equivalent Asp-280 in RNase R may well be the A c c e p t e d M a n u s c r i p t only critical aspartate in this protein. Recent studies have shown that a mutation in aspartate 278 impairs the RNase R activity but the D278N mutant still retains 4% of activity [START_REF] Vincent | The Roles of Individual Domains of RNase R in Substrate Binding and Exoribonuclease Activity: the Nuclease Domain is Sufficient for Digestion of Structured RNA[END_REF]. Moreover, we can say that this residue plays a crucial role in the activity of all the proteins of this family since a mutation in the equivalent residue in RNase R of Legionella pneumophila, Asp-283, also led to the loss the exoribonucleolytic activity of the enzyme [40].

Tyr-324 is responsible for setting the final end-product in RNase R. In RNase II, the RNA molecule is stacked and clamped between two aromatic residues, Tyr-253 and Phe-358 [26] and it was previously shown that Tyr-253 is important for setting the final end-product of 4nts. In the Y253A mutant, the final product released was a 10nts fragment, which is the minimum length necessary for the RNA molecules to establish contacts between the catalytic and the anchoring regions, simultaneously [28]. In RNase R, the equivalent tyrosine is in position 324 (Supplementary Figure 1) and, in this work we wanted to see if this residue also played such an important role in the mechanism of RNA degradation. For that purpose, we introduced the point mutation Tyr324Ala into the pABA-RNR [START_REF] Amblar | The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization[END_REF], and purified the respective protein. The exoribonucleolytic activity was analyzed with different RNA substrates and compared to the wild type. As shown in figure 2a, the mutant Y324A is capable of degrading the poly(A) substrate rendering a final product of 5nts instead of the usual 2nts observed for the wild-type. The same behavior is observed when we used a double stranded 16-30 substrate (Figure 2b). After this last cleavage event, the 5nts resultant fragment is no longer capable to establish the necessary contacts with the protein in order achieve the cleavage of the substrate and is released. As such and similarly to what was shown with the equivalent residue in RNase II, Y253A [28], the corresponding residue in RNase R, Tyr-324, is also the one responsible for the establishment of the final end-product of this protein. When we measured the activity of Y324A, we observed that it retains only 2.5% of activity when compared with the wild type (3.3 ± 0.2 pmol.noml -1 .min -1 and 130.8 ± 6.3 pmol.noml -1 .min -1 , respectively) (Table 3), while in RNase II the equivalent mutation retained approximately 25% of activity [28]. However, when we compared the dissociation constants (K D values) obtained by SPR, the mutant presented a ~6-fold reduction for both substrates tested (Table 4), a similar result to the one obtained for the Y253A mutant in RNase II [28]. These results suggest that the tyrosine residue in RNase R is also important for the binding of the RNA molecule at the 3'-end, but has a much more important function in the activity of the enzyme than in RNase II. This can be due to the fact that the second residue involved in the RNA clamping in RNase II, Phe-358 has no equivalent in RNase R, which can confer to Tyr-324 a more important function in RNA clamping.

The RNB domain of RNase II has catalytic activity and is able to bind to RNA.

When the E. coli RNase II structure was solved it was possible to see that it is constituted by four domains instead of the three initially proposed. This protein is formed by two cold shock domains at the N-terminal region, a central catalytic domain RNB and a S1 domain at the C-terminal region [26]. Before the RNase II structure was known, previous studies were performed with truncated RNase II proteins, where the ΔCSD and RNB mutants included the regions that corresponded to the CSD2 [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF]. In this work we constructed the genuine ΔCSD1+2 and RNB mutants lacking both CSD1/CSD2 and CSD1/CSD2/S1 domains, respectively (Figure 1). We then induced and purified the truncated RNase II proteins and performed activity assays using different substrates. When we analyzed the two truncated RNase II proteins regarding their ability to degrade the single stranded substrate poly(A) we observed that, like RNase II wild type, both RNase II_ΔCSD1+2 and RNase II_RNB were able to degrade the substrate until it reaches 4nts as a final-end product (Figure 3a). However, the activity of these truncated RNase II proteins is very reduced when compared with the wild type enzyme, with RNase II_ΔCSD1+2 retaining only 0.03% of the enzyme's activity and RNase II_RNB less than 0.01% (Table 5). Taking into account that the two CSDs and S1 are the domains responsible for the RNA binding [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF]26], we can clearly say that the loss of activity observed in these mutants is caused by the reduction of their ability to bind to the RNA molecule. The determination of the K D values showed that these mutants are able to bind to RNA molecules less efficiently than the wt enzyme for both substrates tested. While RNase II presents a K D value of 6.5 ± 0.4 nM and 1.3 ± 0.4 nM for 25-mer and poly(A) substrates, respectively [START_REF] Barbas | Determination of key residues for catalysis and RNA-cleavage specificity: one mutation turns RNase II into a "super"-enzyme[END_REF], the absence of both CSDs causes an increase of 4-fold in the K D value, with RNase II_ΔCSD1+2 presenting K D values of 24.1 ± 2.9 nM and 5.2 ± 0.6 nM for the 25-mer and poly(A) substrates, correspondingly (Table 6). The absence of all the RNA binding domains of the protein is responsible for a more pronounced reduction in RNA affinity, with the RNase II_RNB protein presenting almost a 10-fold reduction in the K D values when compared to the wild type enzyme (Table 6). When we analyzed the activity using a double stranded substrate, the 16-30ds, we verified that RNase II_ΔCSD1+2 is able to degrade a few more nucleotides than wt enzyme, releasing fragments with 20-22nts versus the 23-25nts released by RNase II (Figure 3b). This suggests that this mutant can go closer to the double stranded region maybe because the absence of both domains results in less steric hindrance, and allows the better entrance of double stranded molecules into the catalytic cavity. A similar result was obtained with the mutant lacking only the CSD1 [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF]. However, when we tested the same substrate with the RNase II_RNB mutant, we observed that it was capable of degrading all the single stranded 3'overhang of the substrate. The reaction stopped when RNase II_RNB mutant reached the single strand/double strand junction, and released a 16nts blunt-ended fragment (Figure 3b). It is possible to see that the band that corresponds to the 16nts fragment is slightly present in all reactions, even in the control. However, since the intensity of the bands is clearly more pronounced in the RNase II_RNB mutant, and no other products are observed, this indicates that the substrate is being degraded. As such, we can say that the 16nts is the final product that is being released by the mutant. Previous studies have shown that wild type RNase II has more difficulty in degrading the nucleotides adjacent to the double stranded region [START_REF] Vincent | The Roles of Individual Domains of RNase R in Substrate Binding and Exoribonuclease Activity: the Nuclease Domain is Sufficient for Digestion of Structured RNA[END_REF]. Our results confirm that the RNA-binding channel formed by the CSDs and S1 domains in RNase II could be the initial barrier for the degradation of double stranded substrates [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF]. In fact, removal of the RNA-binding domains does allow RNase II to proceed further and the RNase II_RNB mutant protein is able to degrade all the way up to double stranded region.

The cleavage of double stranded RNA is a property of the RNB domain of RNase R.

The structural model of E. coli RNase R indicates that this protein shares a common 3D arrangement with E.coli RNase II and both enzymes have the same domain organization [28]. However, RNase R has an additional HTH (helix-turn-helix) domain at the N-terminal region and a basic region after the S1 domain (Figure 1). It is already known that the CSDs and S1 domains of RNase II are involved in RNA binding and that RNB domain is responsible for the catalytic activity of the enzyme [START_REF] Amblar | Characterization of the functional domains of Escherichia coli RNase II[END_REF]. This modular domain organization has also been postulated for RNase R, and the domains predicted to have the same function [28]. In order to confirm this hypothesis, we constructed truncated RNase R proteins which lack both CSDs and HTH and/or S1 domains as described in figure 1. In order to see the contribution of each domain to the activity and binding of the enzyme, we performed activity assays with a 35-mer poly(A) substrate, calculated the exoribonucleolytic activity and determined the K D values. RNase R is able to degrade single-stranded substrates releasing as final product of the reaction a 2nts fragment [START_REF] Amblar | The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization[END_REF] (Figure 4a). The same behaviour is observed for all the truncated proteins, RNase R_ΔCSD1+2, RNase R_ΔS1 and RNase R_RNB (Figure 4a). However, when we compared the activity of the different enzymes with the wild type, it was possible to verify that all the truncated proteins analysed presented a significant reduction in their activity (Table 7). The absence of both cold shock domains in RNase R_ΔCSD1+2 protein causes a decrease of the activity of the enzyme in more than 1000-fold, from 130.8 ± 6.3 pmol.min - 1 .nmol -1 to 0.2 ± 0.04 pmol.min -1 .nmol -1 . This reduction is more pronounced when the S1 domain is absent, with RNase R_ΔS1 presenting an activity of 0.02 ± 0.002 pmol.min -1 .nmol - 1 . When only the RNB domain is present, the activity of the enzyme decreases more than 10.000-fold, to values lower than 0.01 pmol.min -1 .nmol -1. These results show that both CSD and S1 domains are important for the activity of the enzyme, with S1 domain having a more important role. Since it is postulated that CSDs and S1 domains are involved in RNA binding, the reduction in the activity observed in the truncated proteins can be due to a decrease in RNA binding ability. To confirm this hypothesis, we determined the K D values of each protein by SPR using two different substrates. In fact, all the truncated protein showed a reduction in their K D values when compared to the wild type enzyme for both substrates tested (Table 8). By analyzing the K D values obtained for the truncated proteins, it is possible to see that the CSDs domains are more important for the RNA binding when compared with the S1 domain. This is illustrated by the fact that the RNase R_ΔCSD1+2 protein presented a 3-fold reduction in its binding ability, while the absence of S1 domain was responsible for only a 2-fold reduction (Table 8). When the three domains are absent, the reduction in the ability to bind RNA molecules is much more significative, with RNase R_RNB protein presenting a K D value of 20.7 ± 2.8 nM for the 25-mer substrate and 7.4 ± 0.4 nM for the poly(A) substrate, values that are approximately 6-fold higher than those described for RNase R (Table 8). In RNase R_ΔS1 protein, the reduction in the activity is more pronounced than the verified for the RNase R_ΔCSD1+2 (Table 7), while the dissociation constants obtained for both substrates tested are just 2-fold higher for this protein when compared to wild type versus 3-fold higher for the RNase R_ΔCSD1+2 protein. This result led us to conclude that the CSDs and the S1 domains have different roles in RNA binding. The CSDs must be more important for the recruitment of the substrate, while S1 might help in the orientation and stabilization of the substrate in the catalytic cavity. It is interesting that the RNB domain, per se, is able to bind to RNA, although with a reduction of 6-fold when compared to the wild type. This also helps to explain the residual activity found in the RNase R_RNB protein.

As referred above, RNase R is able to degrade the double stranded substrate 16-30ds releasing a final product of 2nts (Figure 4b) [START_REF] Amblar | The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization[END_REF]. When the truncated RNase R proteins were analysed with the same substrate, we verified that all of them were able to degrade it, releasing the characteristic 2nts fragment (Figure 4b).

RNase R RNA-binding domains discriminate which RNA molecules will be targeted for degradation.

It has been suggested that RNase R needs a 3'-single-stranded overhang at least five nucleotides in length in order to degrade structured RNA molecules [START_REF] Vincent | Substrate recognition and catalysis by the exoribonuclease RNase R[END_REF]. Then cleavage occurs at the single stranded 3' overhang and proceeds processively in the 5' direction, degrading the secondary structure [START_REF] Vincent | Substrate recognition and catalysis by the exoribonuclease RNase R[END_REF][START_REF] Worrall | Information available at cut rates: structure and mechanism of ribonucleases[END_REF]. To verify which domains are responsible for the 3'-overhang requirement in RNase R we performed activity assays with the truncated RNase R proteins using a 16-16ds substrate (which lacks a 3'-overhang). As expected, wild type RNase R is not able to degrade the blunt-ended substrate that was tested (Figure 4c). Previous reports have also shown that the enzyme is not capable of degrading other blunt-ended substrates, even using more extreme reaction conditions (i.e. higher concentrations of enzyme and longer incubation periods) [START_REF] Vincent | Substrate recognition and catalysis by the exoribonuclease RNase R[END_REF]. Surprisingly, the absence of CSDs and/or S1 domains resulted in the ability to degrade the 16-16ds substrate as shown in figure 4c show that the binding domains (CSD1, CSD2 and S1) are blocking the entrance of the double stranded substrates into the catalytic channel. In their absence, the substrate is free to enter into the catalytic cavity and cleavage can occur. Together, these results show that the RNB domain of RNase R is sufficient for the activity of the enzyme. This domain is also capable of binding to RNA substrates, although with less efficiency, and this explains its reduced activity. The decrease in RNA affinity upon deletion of either the CSDs or the S1 domain confirms that these regions indeed function as RNAbinding domains. They are also responsible for the selective degradation of double stranded substrates that contain a 3' single stranded overhang of five or more nucleotides. In fact, both in prokaryotes and eukaryotes, the RNAs targeted for degradation usually contain a short poly(A) tail [START_REF] Houseley | RNA-quality control by the exosome[END_REF]. In case of RNase R, the presence of the binding domains is important for the degradation of only structured RNA molecules that have an overhang of, at least, 5nts length. This suggests that this protein is capable of degrading RNA molecules only when they are targeted to that purpose. As such, the discrimination of which molecules are targeted for decay is made by the CSD1, CSD2 and S1 domains.

In conclusion, the results presented in this study gave an important breakthrough in the understanding of the RNase R mechanism of action. We have identified the Tyr-324 residue that is responsible for setting the final end-product releasing 5nts instead of the usual 2nts observed for the wild-type RNase R. This residue is fully conserved in all domains of life and was proved to have the same function in RNase II. We also show that the Asp-280 residue, present in the RNase R active site, is crucial for the activity of the enzyme but not for RNA binding. We have demonstrated that RNB domains of RNase II and RNase R are able to degrade RNA, even in the absence of the RNA binding domains. The most striking result is that, contrary to the existing literature, the RNB domain of RNase R is sufficient for the degradation of double stranded substrates even in the absence of a 3'-overhang. The RNA binding domains present in the wild type RNase R prevent the degradation of blunt dsRNA molecules. The discrimination of which molecules will be susceptible to degradation by RNase R is made by the CSD1, CSD2 and S1 domains that target the degradation of RNAs that are "tagged" by a 3'-tail. These results can have important implications for the study of poly(A)-dependent degradation mechanisms. A c c e p t e d M a n u s c r i p t

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  . These results 10 Biochemical Journal Immediate Publication. Published on 27 Jul 2009 as manuscript BJ20090839 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090839 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 27 Jul 2009 as manuscript BJ20090839 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090839 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Biochemical Journal Immediate Publication. Published on 27 Jul 2009 as manuscript BJ20090839 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090839 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figure 1 .

 1 Figure 1. Linear representation of the domains of wild type RNase II, RNase R and its derivatives proteins.

Figure 2 .

 2 Figure 2. Exoribonuclease activity of RNase R wild-type and point mutants enzymes.Activity assays were performed as described under "Experimental" using a poly(A) chain of 35 nt (A) or a 30-mer oligoribonucleotide hybridized to the complementary 16mer oligodeoxyribonucleotide, thus obtaining the corresponding double stranded substrate 16-30ds (B). The proteins used and their respective concentrations are shown. The wild-type enzyme was used as control. Samples were taken during the reaction at the time points indicated and reaction products were analyzed in a 20% polyacrylamide/7M urea gel. Control reactions with no enzyme added (Ctrl) were incubated at the maximum reaction time for each protein. Length of substrates and degradation products are indicated in the figure.

Figure 3 .

 3 Figure 3. Exoribonuclease activity of wild-type and truncated RNase II enzymes.Activity assays were performed as described under "Experimental" using a poly(A) chain of 35 nt (a) or a 30-mer oligoribonucleotide hybridized to the complementary 16mer oligodeoxyribonucleotide, thus obtaining the corresponding double stranded substrate 16-30ds (b). The proteins used and their respective concentrations are shown. The wild-type enzyme was used as control. Samples were taken during the reaction at the time points indicated and reaction products were analyzed in a 20% polyacrylamide/7M urea gel. Control reactions with no enzyme added (Ctrl) were incubated at the maximum reaction time for each protein. Length of substrates and degradation products are indicated in the figure.

Figure 4 .

 4 Figure 4. Exoribonuclease activity of wild-type and truncated RNase R enzymes.Activity assays were performed as described under "Experimental" using a poly(A) chain of 35 nt (a), a 30-mer oligoribonucleotide hybridized to the complementary 16mer oligodeoxyribonucleotide, thus obtaining the corresponding double stranded substrate 16-30ds (b) or a 16-mer oligoribonucleotide hybridized to the complementary 16mer oligodeoxyribonucleotide, thus obtaining the corresponding double stranded substrate 16-16ds. The proteins used and their respective concentrations are shown. The wild-type enzyme was used as control. Samples were taken during the reaction at the time points indicated and reaction products were analyzed in a 20% polyacrylamide/7M urea gel. Control reactions with no enzyme added (Ctrl) were incubated at the maximum reaction time for each protein.Length of substrates and degradation products are indicated in the figure.

Figure 1 BiochemicalBiochemical

 1 Figure 1

c

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7

  Biochemical Journal Immediate Publication. Published on 27 Jul 2009 as manuscript BJ20090839 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090839

Table 1 : Plasmids used in this study.

 1

	Tables
	Plasmid	Relevant characteristic	Reference
	pFCT6.1	gene rnb cloned into pET15b, AmpR	Cairrão et al, 2001
	pABA-RNR	gene rnr cloned into pET15b, AmpR	Amblar et al, 2007
	pABA-RNR_D280N	Expresses RNase R where the Asp280 was changed by an Asn	This work
	pABA-RNR_Y324A	Expresses RNase R where the Tyr324 was changed by an Ala	This work
	pFCT_XhoI521	pFCT6.1 with a XhoI restriction site in position 521 of rnb gene	This work
	pABA-RNR_NdeI549	pABA-RNR with a NdeI restriction site in position 549 of rnr gene	This work
	pFCT_ΔCSDs	Expresses RNase II lacking CSD1 and CSD2 domains	This work
	pFCT_RNB	Expresses RNase II lacking CSD1, CSD2 and S1 domains	This work
	pABA-RNR_ΔCSDs	Expresses RNase R lacking HTH, CSD1 and CSD2 domains	This work
	pABA-RNR_ΔS1	Expresses RNase R lacking S1 domain	This work
	pABA-RNR_RNB	Expresses RNase R lacking HTH, CSD1, CSD2 and S1 domains	This work

Table 2 : Primers used in this study.

 2

	Biochemical Journal Immediate Publication. Published on 27 Jul 2009 as manuscript BJ20090839

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Table 3 : Exoribonucleolytic activity of wild-type RNase R and point mutant enzymes.

 3 Exoribonucleolytic activity was assayed using a 35nt poly(A) chain as substrate. Activity assays were performed in triplicate as described in "Experimental".

	Protein	(	Protein Activity

pmol.min -1 .nmol -1 ) Relative Activity (%)

	wt RNase R	130.8 ± 6.3	100
	D280N	<<0.01	<<0.01
	Y324A	3.3 ± 0.2	2.5

Table 4 : RNA binding affinity of wild-type RNase R and point mutant enzymes.

 4

	The dissociation constants (K D ) were determined by Surface Plasmon Resonance using
	Biacore 2000 with a 25-nt RNA oligomer (5'-Biotin-CCC GAC ACC AAC CAC UAA AAA
	AAA A-3') and 30nts poly(A) as substrates as described in "Experimental".
	Protein	K D 25-mer	Relative K D 25-mer	K D poly(A)	Relative K D poly(A)
	wt RNase R	3.2 ± 0.4	1.0	1.2 ± 0.1	1.0
	D280N	2.2 ± 0.3	0.7	1.3 ± 0.2	1.1
	Y324A	20.4 ± 1.4	6.4	6.7 ± 0.3	5.6

Table 5 : Exoribonucleolytic activity of wild-type and truncated RNase II proteins.

 5 Exoribonucleolytic activity was assayed using a 35nt poly(A) chain as substrate. Activity assays were performed in triplicate as described in "Experimental".

	Protein	Protein Activity (pmol.min -1 .nmol -1 )	Relative Activity (%)
	wt RNase II	299.4 ± 36.0	100
	RNase II_ΔCSD	0.1 ± 0.01	0.03
	RNase II_RNB	<<0.01	<<0.01

Table 6 : RNA binding affinity of wild-type and truncated RNase II proteins.

 6

	The dissociation constants (K D ) were determined by Surface Plasmon Resonance using
	Biacore 2000 with a 25-nt RNA oligomer (5'-Biotin-CCC GAC ACC AAC CAC UAA AAA
	AAA A-3') and 30nts poly(A) as substrates as described in "Experimental".
	Protein	K D 25-mer	Relative K D 25-mer	K D poly(A)	Relative K D poly(A)
	wt RNase II	6.5 ± 0.4	1.0	1.3 ± 0.4	1.0
	RNase II_ΔCSD	24.1 ± 2.9	3.7	5.2 ± 0.6	4.0
	RNase II_RNB	59.9 ± 6.0	9.2	11.7 ± 2.1	9.0

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Table 7 : Exoribonucleolytic of wild-type and truncated RNase R proteins.

 7 Exoribonucleolytic activity was assayed using a 35nt poly(A) chain as substrate. Activity assays were performed in triplicate as described in "Experimental".

	Protein	(	Protein Activity

pmol.min -1 .nmol -1 ) Relative Activity (%)

	wt RNase R	130.8 ± 6.3	100
	RNase R_ΔCSD	0.2 ± 0.04	0.1
	RNase R_ΔS1	0.02 ± 0.002	0.02
	RNase R_RNB	<<0.01	<<0.01

Table 8 : RNA binding affinity of wild-type and truncated RNase R proteins.

 8 The dissociation constants (K D ) were determined by Surface Plasmon Resonance using Biacore 2000 with a 25-nt RNA oligomer (5'-Biotin-CCC GAC ACC AAC CAC UAA AAA AAA A-3') and 30nts poly(A) as substrates as described in "Experimental".

	Protein	K D 25-mer	Relative K D 25-mer	K D poly(A)	Relative K D poly(A)
	wt RNase R	3.2 ± 0.4	1.0	1.2 ± 0.1	1.0
	RNase R_ΔCSD	9.6 ± 1.4	3.0	3.1 ± 0.1	2.6
	RNase R_ΔS1	5.4 ± 0.3	1.7	2.1 ± 0.2	1.8
	RNase R_RNB	20.7 ± 2.8	6.5	7.4 ± 0.4	6.2

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgments

We thank Miguel Luís for helping with the images. Rute G. Matos was a recipient of a PhD fellowship and A. Barbas was a recipient of a Post Doctoral fellowship, both of them funded by FCT-Fundação para a Ciência e a Tecnologia, Portugal. This work was supported by FCT-Fundação para a Ciência e a Tecnologia, Portugal.