RNA Binding Activity of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit from Chlamydomonas reinhardtii

Abstract

Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289–16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5′-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG. Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289–16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5′-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG. When plants and algae absorb light energy that exceeds the level of electron carrier saturation they generate reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RB, RNA binding; EMSA, electrophoretic mobility shift assays; SK, pBluescript; RBD, RNA binding domain; LSU, large subunit; UTR, untranslated region; DTT, dithiothreitol; aa, amino acids. that cause a variety of cellular and molecular damage. This phenomenon is referred to as photoinhibition and is common to all photosynthetic organisms (1Barber J. Andersson B. Trends Biochem. Sci. 1992; 17: 61-66Abstract Full Text PDF PubMed Scopus (843) Google Scholar, 2Osmond C.B. Baker N.R. Boyer J.R. Photoinhibition of Photosynthesis, from Molecular Mechanisms to the Field. BIOS Scientific Publishers Ltd., Oxford1994: 1-24Google Scholar, 3Prásil O. Adir N. Ohad I. Barber J. The Photosystems: Structure, Function, and Molecular Biology. Elsevier Science Publishers B. V., Amsterdam1992: 295-348Crossref Google Scholar). Recovery from photoinhibition can be achieved by decreasing the chlorophyll content and by activating a variety of antioxidant pathways that involve ascorbate and glutathione (4Foyer C.H. Lopez-Delgado H. Dat J.F. Scott I.M. Physiol. Plant. 1997; 100: 241-254Crossref Google Scholar, 5Noctor G. Gomez L. Vanacker H. Foyer C.H. J. Exp. Bot. 2002; 53: 1283-1304Crossref PubMed Scopus (689) Google Scholar). Ribulose-1,5-bisphosphate carboxylase (Rubisco) is the key enzyme in photosynthetic carbon assimilation. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family. Assembly of the holoenzyme is mediated by the chloroplast chaperonins cpn60 and cpn10 (6Gutteridge S. Gatenby A.A. Plant Cell. 1995; 7: 809-819Crossref PubMed Scopus (139) Google Scholar, 7Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (329) Google Scholar). We previously showed that transfer of the green algae C. reinhardtii from low light (70 μmol m–2 s–1) to high light (700 μmol m–2 s–1) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco (8Shapira M. Lers A. Heifetz P. Yrihimovitz V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). These light-induced effects were found to be transient, with cell recovery taking place within 6–12 h once chlorophyll levels were reduced and ROS levels were decreased. It was further found that translation of Rubisco LSU varies with the changes in ROS production and correlates with alterations in the ratio between oxidized and reduced glutathione. Upon transfer to high light the glutathione pool shifts to its oxidized form, and LSU synthesis stops almost completely. When the cells recover from light stress, the glutathione pool shifts back to its reduced form, and LSU translation resumes (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Rubisco holoenzyme is highly susceptible to oxidative stress in vivo. Excess ROS caused a rapid translocation of the soluble enzyme complex into the chloroplast membrane and the formation of intermolecular cross-linking between the large subunits via disulfide bonds (10Mehta R.A. Fawcett T.W. Porath D. Mattoo A.K. J. Biol. Chem. 1992; 267: 2810-2816Abstract Full Text PDF PubMed Google Scholar). Furthermore, oxidative stress can cause direct fragmentation of Rubisco LSU at Gly-329 into 37- and 16-kDa polypeptides in illuminated intact chloroplasts in chloroplast extracts and in its purified form when exposed to a hydroxyl radical-generating system (11Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Translational regulation allows plants to respond quickly to environmental changes such as light intensities and is, therefore, a predominant mechanism in chloroplasts. Upstream UTRs are expected to play a key role in translation of chloroplast genes via interaction with regulatory proteins. A group of RNA binding (RB) proteins with molecular masses of 60, 55, 47, and 38 kDa the of the RNA A. J. PubMed Scopus Google Scholar). a high with the protein C.B. A. A. S. Sci. S. A. PubMed Scopus Google and high to protein disulfide J. 1997; PubMed Scopus Google Scholar, T. O. A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Binding of the protein complex is mediated by and light-induced of It was that as a redox that binding of T. A. A. A. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). with molecular masses the of and chloroplast Gillham N.W. Boynton J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google it is that RNA-binding proteins of 47 kDa S. Plant Mol. Biol. 2002; PubMed Scopus Google Scholar). In an to the mechanism that the of regulation Rubisco LSU, we examined how the redox of RNA-binding proteins affected interaction with the rbcL We that the interaction between RNA-binding proteins and the rbcL is interrupted by oxidative stress and that Rubisco LSU can bind RNA in its oxidized form, in a manner. and reinhardtii cells were in high reduced with of and at were illuminated with light μmol m–2 s–1) using The an at the that translation of Rubisco LSU at M. M. Sci. S. A. PubMed Google and was at under conditions. Rubisco cells were in under low light conditions (70 μmol m–2 in RNA of rbcL from C. reinhardtii was from a that the of chloroplast M. M. J. Mol. Biol. PubMed Scopus Google Scholar). of the was into at the in The of rbcL to to the was from using the and The was into the and between the and were in The rbcL was by and synthesis of the and In vitro synthesis of the from and nuclear was from and was the of A. and were from the Chlamydomonas at was with and and were with In vitro synthesis of the from the was with RNA of a RNA was using as a The was by and RNA was with RNA of reinhardtii cells were at and protein extracts were as previously A. J. PubMed Scopus Google Scholar, Gillham N.W. Boynton J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were in high reduced and at a of The were in and at were in of low and in the of and The cells were by a at The cells were at in an The were and further at h at in an The was and a at a of The was with of high DTT, and and with and The bound proteins were with a of in low were DTT, and and at The were by and protein content was by In RNA RNA were in vitro using of in DTT, of and of and of or RNA in a of The were at by the of of and were an at The were from the a of in these conditions were to that between and of RNA. that were used were as that the were to and all the were at RNA were to production of a and to its and mobility shift were as previously Gillham N.W. Boynton J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). from the of were at with of in in a of The were to the RNA in the of of in a of an of at of and were and the were a in and conditions were The were in a of and and to In experiments RNA were in of and with the protein the of the were as previously Gillham N.W. Boynton J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins from the of purified Rubisco or the recombinant polypeptides that to Rubisco LSU or its and a were at with of in in a of RNA was to the protein with of in a of of at the binding were and by at in a RNA were with of A at The were were by and to or by In experiments RNA were in and with the protein the of the how the redox affected the binding or was to the protein extracts or to the purified Rubisco the of the two-dimensional was as previously J. Biol. Chem. Full Text PDF PubMed Google Scholar). the was by using that between and the was by Rubisco holoenzyme from were a of T. J. from the were one- and two-dimensional and a membrane was using and a of protein A with binding was with and in a and Rubisco C. reinhardtii cells were in to and in a as The cell was a in low and was by a of in low The was low and a in low The was h at in a at and were of were examined by using of LSU and in The region that Rubisco LSU was using from of the rbcL the was and the was The region the amino terminus of Rubisco LSU was by using the and the The region that the domain of Rubisco was by using the and the that and were and are in The were into the P. S. 1999; PubMed Scopus Google Scholar). was in cells by of the with at The were once in in a and and in a using The was at at The protein in the soluble was a the recombinant domain was it was a previously J. Molecular A and by The soluble recombinant polypeptides were to using of Rubisco of the large subunit of Rubisco were the that a structural of proteins T. C. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). RNA Binding of to the rbcL and to the translational arrest of the rbcL at high light and oxidative stress correlated with changes in the redox of proteins that bind to its electrophoretic mobility shift were The of proteins that bind was by of cell extracts a by with a of RNA from to to the translational of the rbcL gene was in vitro and with the protein Binding of proteins to the RNA was by of the RNA in as with RNA. RNA binding activity was with the of rbcL of the RNA-protein in that the binding a Binding to the of rbcL was interrupted in a by of the rbcL Binding was affected by the of of RNA or by a large of and the RNA the RNA-protein In binding was with a RNA of from the between the shift of glutathione to its oxidized form light stress and the translational arrest of Rubisco LSU (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google binding were under oxidizing and reducing conditions. proteins of binding to the of rbcL in were with of and and the of the rbcL shifting of the RNA was in the of and was by and and the binding activity These that the redox of RNA-binding to interact with the of the rbcL Binding of to the of of the proteins that bind to the rbcL was using RNA to the of rbcL was with proteins from the and to by we found that proteins with a molecular of 81, 62, 51, 47, and kDa bind to the of rbcL. that the binding was to the of cross-linking was in the of RNA Binding of the and proteins was by the rbcL and was affected by the RNA or a the RNA-protein binding of the and proteins was by the rbcL interaction with the RNA was of to the of rbcL to the of how the redox of binding of proteins to the of UV-cross-linking were in the of reduced and oxidized glutathione. extracts from the were with of the of the rbcL oxidizing conditions binding of the and proteins cross-linking of a new 55-kDa protein was In of the molecular of protein and the LSU of Rubisco, the that an was Rubisco LSU an RNA-binding protein was one- and two-dimensional with were with cross-linking with the of rbcL. The proteins were by two-dimensional and The were exposed to a and and with an Rubisco LSU A and of the and that the 55-kDa protein that to the of rbcL under oxidizing conditions with Rubisco LSU two-dimensional binding of Rubisco LSU to the rbcL under oxidizing conditions was by using the purified enzyme in UV-cross-linking shown in A and the purified protein with the rbcL in direct with the binding was in the of GSSG. In the 55-kDa protein was in UV-cross-linking experiments under oxidizing conditions using M. M. Sci. S. A. PubMed Google a that to Rubisco LSU to a that translation the binding between Rubisco LSU and its rbcL were with that to the rbcL in its and of binding to the rbcL was with of that in vitro the binding of Rubisco LSU to its was sequence-independent A was from experiments that the between RNA and the rbcL of these RNA the binding between Rubisco LSU and its RNA with therefore, appears that under oxidizing conditions Rubisco LSU binds to RNA. In the of a large of a was from that in protein by RNA further the RNA binding of Rubisco LSU in the of UV-cross-linking experiments were with protein extracts from the and with purified Rubisco using RNA A binding was chloroplast RNA and under reducing conditions. under oxidizing the 55-kDa Rubisco LSU bound to the purified Rubisco LSU with all the RNA the of rbcL in the and the of and nuclear and the RNA These that oxidized Rubisco LSU interact with RNA in a sequence-independent manner. the interaction of oxidized LSU with RNA was cross-linking was with The enzyme was in cross-linking with the rbcL When Rubisco was oxidized by the of and binding to RNA was affected to However, Rubisco was and oxidized by as low as affected its to bind RNA. when the protein was and cross-linking a molecular species that from cross-linking between subunits The proteins were Rubisco was oxidized and A of is that interact to form that the domain that binds exposure to The RNA Binding of Rubisco the structural the RNA binding activity in Chlamydomonas Rubisco LSU, we to from its crystal structure A. Andersson I. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. H. J. N. T. A. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). The crystal structure of Rubisco of a domain and a amino terminus domain The was that the RNA binding of Rubisco is to structural with RNA binding proteins. a that a structural of proteins T. C. J. Mol. Biol. 1995; PubMed Scopus Google we were to structural similarity between the amino terminus of Rubisco and a of RNA-binding proteins Struct. Biol. PubMed Scopus Google Scholar). the LSU amino terminus and these RNA-binding proteins the structure as the structural similarity between the RNA binding domain of the in complex with the A. Andersson I. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google and the amino terminus of Rubisco LSU PubMed Scopus Google Scholar). The of of the structure is the amino such similarity was found between the domain of LSU and structure of an RNA-binding we that the RNA binding of the LSU is within its amino-terminal further the amino terminus of Rubisco LSU can bind the gene protein domain was and in The binding of the Rubisco holoenzyme and the LSU to RNA was by cross-linking with protein in the of RNA The of Rubisco holoenzyme was the of rbcL and RNA. The of the recombinant polypeptide RNA was The RNA binding by Rubisco and its amino terminus were with the RNA-binding proteins M. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of binding to RNA was examined the recombinant LSU and the protein to amino and The purified recombinant polypeptides with The amino terminus in to that within the The recombinant LSU to RNA in the of as with the holoenzyme However, binding of the amino-terminal domain with and without GSSG. When protein was in it was RNA The of The domain showed binding of RNA in the or of GSSG. of the crystal structure of Rubisco A. Andersson I. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. H. J. N. T. A. J. Mol. Biol. 2002; PubMed Scopus Google that the amino terminus is exposed to the surface of the protein and is by the structure and However, the amino-terminal domain is the RNA binding it be exposed to the oxidative stress leads to structural changes in Rubisco H. A. M. J. PubMed Scopus Google V. and M. Shapira, in these in exposure of the amino terminus domain, it to RNA. Assembly of protein the of chloroplast genes is a key in translational regulation A. Plant Physiol. 1997; PubMed Scopus Google Scholar, Plant Mol. Biol. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). that the interaction between RNA-binding proteins and the of are to regulation by the redox of the electron T. A. A. A. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, A. Plant Physiol. 1997; PubMed Scopus Google Scholar). rbcL a regulatory that leads to translational arrest of the large subunit of Rubisco light-induced oxidative the redox of the electron in the photosynthetic translation of Rubisco LSU to of ROS in the chloroplast (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). oxidative stress caused a shift in the glutathione pool toward its oxidized form, and that translation of Rubisco LSU was the cells reduced chlorophyll content and from the level of ROS and the ratio was In an to of we potential regulatory RNA-protein interactions that were affected by the redox of glutathione. EMSA, we that the RNA-binding proteins the of the rbcL The of binding was by using the rbcL and RNA UV-cross-linking experiments with the of rbcL a group of proteins that bound under reducing with molecular masses of 81, 62, 51, 47, and However, binding of the and proteins was by the RNA and by such as RNA or binding A of binding was the of and that these proteins bound to chloroplast These are in with proteins 81, 62, 51, 47, and kDa that interact with the rbcL as as with chloroplast and Gillham N.W. Boynton J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In a S. Plant Mol. Biol. 2002; PubMed Scopus Google using that membrane proteins of 60, 47, and to and binding by light and by of the The potential role of changes in the redox of in binding of proteins to the rbcL was by and UV-cross-linking experiments under oxidizing and reducing conditions. of the binding activity of proteins to the rbcL as with binding under reducing conditions. These that of the redox potential translation of Rubisco LSU by the redox of RNA-binding proteins. UV-cross-linking experiments showed that binding of the and proteins was interrupted under oxidizing and a new protein of 55 kDa that with Rubisco LSU was to the as in one- and two-dimensional of the 55-kDa protein in with and saturation within the G. Gomez L. Vanacker H. Foyer C.H. J. Exp. Bot. 2002; 53: 1283-1304Crossref PubMed Scopus (689) Google further the of the 55-kDa UV-cross-linking experiments were with purified Rubisco under and oxidizing conditions. LSU from purified Rubisco bound to its under oxidizing conditions In the protein was in extracts from a that to Rubisco However, experiments that the binding was and from the of rbcL the binding of purified Rubisco LSU to its and a was with RNA. Furthermore, we found that under oxidizing purified Rubisco LSU bound to a variety of RNA from chloroplast and nuclear genes as as to a RNA of protein subunits that into in the chloroplast is to that the level of the The terminus of the subunit with the of gene and its translation J. Sci. S. A. PubMed Scopus Google Scholar). was Rubisco When of the small subunit of Rubisco was by in C. reinhardtii S. J. C.H. L. Sci. S. A. PubMed Scopus Google or by of plants with rbcS synthesis of the LSU was L. M. Scopus Google Scholar). The mechanism that of Rubisco LSU in the of the eight small subunits However, an LSU its the of eight small subunits be translation of LSU S. J. C.H. L. Sci. S. A. PubMed Scopus Google Scholar). In we that Rubisco LSU the structural of an RNA-binding an be binding of RNA in vitro in a sequence-independent manner. we the that in Rubisco LSU in with protein a binding that the translational binding was the in in the of group by a RNA interaction with RNA is mediated by an RNA RNA that in the RNA S. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the binding of proteins to chloroplast the under reduced and oxidizing conditions. However, the of a regulatory in with the role of that of chloroplast is synthesis of in C. it with high molecular is a direct interaction between and its RNA J. J. 2000; PubMed Scopus Google Scholar). are the proteins of J. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar, J. J. Biol. 2002; PubMed Scopus Google and is of the chloroplast gene M. Sci. S. A. 2000; PubMed Scopus Google Scholar). The crystal structure of Rubisco previously A. Andersson I. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. H. J. N. T. A. J. Mol. Biol. 2002; PubMed Scopus Google RNA binding were The large subunit of the protein of a domain and a amino terminus domain that was as a domain by T. C. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). The amino terminus is to a structure by a of RNA-binding proteins the RNA binding domain The is composed of a structure that a the Struct. Biol. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar, H. G. 1997; 7: PubMed Scopus Google Scholar). The RNA binds to the of the The to the proteins that the binding is the between the amino terminus domain and the of the terminus domain the binding at the the formation of disulfide bonds stress region and it to RNA. A role is the of buried within the holoenzyme. These to redox regulation of Rubisco H. A. M. J. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of be at A recombinant polypeptide that the was of binding RNA in with a binding that was to that with purified Rubisco and in with RNA binding proteins M. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. C.B. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, binding of the recombinant amino-terminal domain the redox in the binding in with that under reducing conditions interaction with the RNA is by of the The role the RNA binding activity of Rubisco is it a in an by protein its RNA a such as polypeptides C. J. S. P. 2000; PubMed Scopus Google or the stress D. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google can bind RNA in a sequence-independent manner. It was that as an RNA that binds to RNA This binding the formation of that the RNA susceptible to it was that that involve and translation B. M. Sci. S. A. 2000; PubMed Scopus Google Scholar). We that Rubisco LSU play a It was previously that oxidative stress, Rubisco from the into the chloroplast translation of chloroplast proteins place J. Biol. PubMed Scopus Google Scholar). it is that oxidized Rubisco LSU as an RNA G. Mol. Biol. 2002; PubMed Google that RNA from that oxidative We T. J. from at Rubisco, A. from the of Science at and L. from the Chlamydomonas at Chlamydomonas and and