Nsa2 Is an Unstable, Conserved Factor Required for the Maturation of 27 SB Pre-rRNAs

In Saccharomyces cerevisiae, a large variety of pre-ribosomal factors have been identified recently, a number of which are still of unknown function. The essential pre-ribosomal 30-kDa protein, Nsa2, was characterized as one of the most conserved proteins from yeast to human. We show here that the expression of the human orthologue TINP1 complements the repression of NSA2 in yeast. Nsa2 was co-purified in several pre-ribosomal complexes and found to be essential for the large ribosomal subunit biogenesis. Like several other factors of the pre-60 S particles, the absence of Nsa2 correlated with a decrease in the 25 S and 5.8 S ribosomal RNA levels, and with an accumulation of 27 SB pre-ribosomal RNA intermediates. We show that Nsa2 is a functional partner of the putative GTPase Nog1. In the absence of Nsa2, Nog1 was still able to associate with pre-ribosomal complexes blocked in maturation. In contrast, in the absence of Nog1, Nsa2 disappeared from pre-60 S complexes. Indeed, when ribosome biogenesis was blocked upstream of Nsa2, this short half-lived protein was largely depleted, suggesting that its cellular levels are tightly regulated. In Saccharomyces cerevisiae, a large variety of pre-ribosomal factors have been identified recently, a number of which are still of unknown function. The essential pre-ribosomal 30-kDa protein, Nsa2, was characterized as one of the most conserved proteins from yeast to human. We show here that the expression of the human orthologue TINP1 complements the repression of NSA2 in yeast. Nsa2 was co-purified in several pre-ribosomal complexes and found to be essential for the large ribosomal subunit biogenesis. Like several other factors of the pre-60 S particles, the absence of Nsa2 correlated with a decrease in the 25 S and 5.8 S ribosomal RNA levels, and with an accumulation of 27 SB pre-ribosomal RNA intermediates. We show that Nsa2 is a functional partner of the putative GTPase Nog1. In the absence of Nsa2, Nog1 was still able to associate with pre-ribosomal complexes blocked in maturation. In contrast, in the absence of Nog1, Nsa2 disappeared from pre-60 S complexes. Indeed, when ribosome biogenesis was blocked upstream of Nsa2, this short half-lived protein was largely depleted, suggesting that its cellular levels are tightly regulated. Ribosome biogenesis is a highly conserved process among eukaryotes and results in the synthesis of functional small and large ribosomal subunits, necessary for the translation of mRNAs into proteins in the cytoplasm. This essential process is tightly regulated; indeed, in exponentially growing Saccharomyces cerevisiae cells, it accounts for about 60% of the metabolic effort (1Warner J.R. Trends Biochem. Sci. 1999; 24: 437-440Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar), whereas it is almost completely turned off during the stationary phase. The pathway begins with the transcription by RNA polymerase I of a 35 S ribosomal RNA (rRNA) precursor and of the 5 S rRNA by RNA polymerase III. This transcription, together with the nuclear import of ribosomal proteins, pre-ribosomal factors, and small nucleolar RNAs, is responsible for the self-assembly of the nucleolus (reviewed in Ref. 2Dundr M. Misteli T. Biochem. J. 2001; 356: 297-310Crossref PubMed Scopus (330) Google Scholar), a region of the nucleus specialized in the production of ribosomes. Association of ribosomal proteins and pre-ribosomal factors with nascent pre-rRNAs gives birth to a 90 S pre-ribosomal complex, which undergoes various steps of maturation, first in the nucleolus, then in the nucleoplasm, and finally in the cytoplasm after export through the pores of the nuclear envelope (for a review of the whole pathway, see Refs. 3Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (655) Google Scholar, 4Fromont-Racine M. Senger B. Saveanu C. Fasiolo F. Gene. 2003; 313: 17-42Crossref PubMed Scopus (482) Google Scholar, 5Tschochner H. Hurt E. Trends Cell Biol. 2003; 13: 255-263Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Along this maturation, the 90 S complex separates into a pre-60 S complex, which will generate the large ribosomal subunit containing mature 25 S, 5.8 S, and 5 S rRNAs, and a pre-40 S complex, which will generate the small ribosomal subunit containing 18 S rRNA. A large number of factors are necessary for the correct modification, cleavage, and processing of pre-rRNAs, the positioning of ribosomal proteins, and the export of the pre-60 S and pre-40 S particles toward the cytoplasm. More than 100 factors associated with pre-60 S complexes (pre-60 S factors) have been identified to date, a number of which remain to be characterized (4Fromont-Racine M. Senger B. Saveanu C. Fasiolo F. Gene. 2003; 313: 17-42Crossref PubMed Scopus (482) Google Scholar, 6Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4010) Google Scholar, 7Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (292) Google Scholar, 8Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. Nature. 2002; 415: 180-183Crossref PubMed Scopus (3086) Google Scholar, 9Krogan N.J. Peng W.T. Cagney G. Robinson M.D. Haw R. Zhong G. Guo X. Zhang X. Canadien V. Richards D.P. Beattie B.K. Lalev A. Zhang W. Davierwala A.P. Mnaimneh S. Starostine A. Tikuisis A.P. Grigull J. Datta N. Bray J.E. Hughes T.R. Emili A. Greenblatt J.F. Mol. Cell. 2004; 13: 225-239Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Some of them display obvious enzymatic functions (RNases, helicases, methylases, etc.). Others are annotated as putative enzymes based on sequence similarities, but their exact role in the biogenesis is still unclear. Among these factors, a set of putative GTPases is required at various steps of the maturation of the large subunit, namely nuclear-nucleolar Nog1, Nog2, Nug1, and cytoplasmic Lsg1 (10Bassler J. Grandi P. Gadal O. Lessmann T. Petfalski E. Tollervey D. Lechner J. Hurt E. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Saveanu C. Bienvenu D. Namane A. Gleizes P.E. Gas N. Jacquier A. Fromont-Racine M. EMBO J. 2001; 20: 6475-6484Crossref PubMed Scopus (157) Google Scholar, 12Kallstrom G. Hedges J. Johnson A. Mol. Cell. Biol. 2003; 23: 4344-4355Crossref PubMed Scopus (117) Google Scholar). As putative GTPases, these proteins are believed to participate in the control of biogenesis; they may play a part as proofreaders of the correct maturation of pre-ribosomal particles at a precise step. In a previous work, we demonstrated that the physical and functional partners Nog1 and Rlp24 are required for adequate processing of the internal transcribed spacer 2 (ITS2) 3The abbreviations used are: ITS2, internal transcribed spacer 2; 3-AT, 3-aminotriazol; G6PDH, glucose-6-phosphate dehydrogenase; ORF, open reading frame; TAP, tandem affinity purification; TEV, nuclear inclusion A protein of tobacco etch virus; YPD, yeast extract-peptone-d-glucose; snRNA, small nuclear RNA. in 27 S pre-rRNAs, located between the 5.8 S and 25 S mature rRNAs (13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google Scholar). Here, we have focused on a partner of Nog1, the essential pre-60 S factor, Nsa2 (for Nop seven-associated 2), which was found associated with pre-60 S complexes in tandem affinity purifications (6Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4010) Google Scholar, 7Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (292) Google Scholar, 13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google Scholar, 14Harnpicharnchai P. Jakovljevic J. Horsey E. Miles T. Roman J. Rout M. Meagher D. Imai B. Guo Y. Brame C.J. Shabanowitz J. Hunt D.F. Woolford Jr., J.L. Mol. Cell. 2001; 8: 505-515Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). It contains an S8 domain, named after the Rps8 archaeal and eukaryotic ribosomal proteins, which could be in This to be one of the most conserved proteins in the eukaryotic F. E. S. R. B. N. R. G. G. 2001; PubMed Scopus Google Scholar). with as we show here that the human orthologue TINP1 is able to the repression of NSA2 in yeast. The absence of Nsa2 results in S levels in yeast P. Jakovljevic J. Horsey E. Miles T. Roman J. Rout M. Meagher D. Imai B. Guo Y. Brame C.J. Shabanowitz J. Hunt D.F. Woolford Jr., J.L. Mol. Cell. 2001; 8: 505-515Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). We show in this that this protein is required for the of the pre-60 S complexes through the and processing the protein levels of Nsa2 to be tightly and on S biogenesis upstream in the metabolic and yeast used in the are in by to A. O. A. F. C. 21: PubMed Scopus Google Scholar). these may be in the with the from the of A. E. J. P. PubMed Scopus Google Scholar). for by in and of by D. R. R. 8: PubMed Scopus Google Scholar). in the control of the and The was by the TINP1 open reading from the TINP1 in the control of the NSA2 and The sequence of used for and as (13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google used in this G. A. B. M. Mann M. Seraphin B. 1999; PubMed Scopus Google A. E. J. P. PubMed Scopus Google A. E. C. F. 13: PubMed Scopus Google A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4010) Google C. Bienvenu D. Namane A. Gleizes P.E. Gas N. Jacquier A. Fromont-Racine M. EMBO J. 2001; 20: 6475-6484Crossref PubMed Scopus (157) Google in a and with a yeast in The on at that their when with the with an was and The by of the and the by in steps on at and with was with the with the in on and and then on and for the of The of the was by on containing 25 RNA and with and to on on and then to and with various with to with and the then on with and protein from exponentially growing yeast as C. Bienvenu D. Namane A. Gleizes P.E. Gas N. Jacquier A. Fromont-Racine M. EMBO J. 2001; 20: 6475-6484Crossref PubMed Scopus (157) Google and on by for at in a In of the the proteins with on and to affinity proteins with a of the complex proteins by as at to and as by Saveanu (13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google Scholar). by of with protein in and by of with and and from J. L. Woolford of F. and F. Fasiolo was from from used at a of the was with the of complexes associated with to the G. A. B. M. Mann M. Seraphin B. 1999; PubMed Scopus Google Scholar), from of yeast The protein by and by as the of RNAs, the first of the was with a containing complex and a containing The in the with and then by as of various pre-60 S the control of a from to The at was for and the a to at proteins from these the and by as Nog1 and Nsa2 and is a putative GTPase in the biogenesis of the S ribosomal In a previous (13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google Scholar), we that Nog1 is together with for adequate of in the rRNA maturation pathway, suggesting that it be a of the through this step. into the of Nog1 in this we for functional partners of this of by on the whole in a the on a to a as as 27 SB processing when at The most was for which for at 25 and but pre-60 S export when for 2 at We a with these at In to the was in of which the ORF, The was by a from upstream of the NSA2 to of its NSA2 and with their and in 2 on the of the the of the for at and and at 25 and when NSA2 was In to NSA2 the between NSA2 and is to a functional of the from this between we could through a a physical between Nog1 as a and Nsa2 as a This of the on containing to 5 Nog1 and Nsa2 with in the for of 25 As a was between the Rlp24 and the Nsa2 whereas this could with its partner Nog1 this 25 A was between Nsa2 and of Nog1 blocked in a in a as as with a of Nog1 to its first a conserved located upstream of the of the protein the of Nsa2 in protein is a protein, which was identified in pre-60 S complexes by tandem affinity P. Jakovljevic J. Horsey E. Miles T. Roman J. Rout M. Meagher D. Imai B. Guo Y. Brame C.J. Shabanowitz J. Hunt D.F. Woolford Jr., J.L. Mol. Cell. 2001; 8: 505-515Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). This protein is highly conserved among its human nuclear protein and with Nsa2, which it one of the most conserved proteins in the yeast F. E. S. R. B. N. R. G. G. 2001; PubMed Scopus Google Scholar). The of Nsa2 in this We was the orthologue of Nsa2 by A 2 TINP1 the control of the was able to a in which NSA2 was a in with the with the In contrast, in a the of TINP1 was that TINP1 is the orthologue of 27 SB rRNA in the of functional between Nog1 and Nsa2 that factors be in the cellular previous P. Jakovljevic J. Horsey E. Miles T. Roman J. Rout M. Meagher D. Imai B. Guo Y. Brame C.J. Shabanowitz J. Hunt D.F. Woolford Jr., J.L. Mol. Cell. 2001; 8: 505-515Abstract Full Text Full Text PDF PubMed Scopus (256) Google that Nsa2 is required for of adequate cellular S ribosomal subunit these that Nsa2 be required for the of the large ribosomal subunit biogenesis. this a in which NSA2 was control of a was to from to rRNAs and and this repression This a decrease in the levels of mature 5.8 S and 25 S rRNA in the with the in the of 18 S rRNA levels As as rRNA are we a in the S levels to which correlated with an in the 27 We that Nsa2 is required for and processing steps of during the maturation of the large ribosomal subunit 27 SB and S rRNA in the of Nsa2 in the processing of ITS2, we rRNA in complexes. affinity on of a a as a and on the 27 S, and S rRNA in the complexes when with the the and the of the be found in complexes We could a of 5 S rRNA in the with this RNA polymerase associated with pre-60 S particles when of In contrast, between was in the levels of the other pre-rRNAs 35 mature RNA polymerase I S, 5.8 S, 18 The as as the levels, to that Nsa2 is in pre-60 S complexes during the Nog1 in S the in which Nog1 and Nsa2 are we to them in the of the We first that proteins in particles in by and could be in to the S As a the ribosomal protein was found to in the S the S and with the of the we to one of these factors was essential for the of the other one on the of complexes was in a to for in to these Nsa2 was in the S and was in This correlated with a of the complex the ribosomal protein was still in the S S and on we that Nog1 is essential for the of Nsa2 in pre-60 S In contrast, still with S particles when NSA2 was for the absence of Nsa2 the of Nog1 to pre-60 S complexes. biogenesis of the large ribosomal subunit was rRNA maturation was and a decrease in the S levels was on these that Nog1 is upstream of Nsa2 in the process of ribosome biogenesis. the S biogenesis was we that the of complexes in absence of Nsa2 could a of the large ribosomal subunit at a precise in the of the S the of Nsa2 to be in pre-ribosomal complexes than Nog1, we that we the biogenesis of Nog1 by NSA2 and in this yeast in pre-ribosomal complexes of the step. particles by the from a for Nsa2 a in which NSA2 was in between complexes by for pre-60 S factors proteins of the large ribosomal subunit NSA2 was we in the complexes a in the of and and a of and Rlp24 levels between the and the complexes. As the Nog1 and the large ribosomal protein found in in complexes. these that Nsa2 on the large ribosomal subunit biogenesis after the of Nog1, and to pre-60 S particles and the of Nog2, and levels for Rlp24 that this factor, is in in complexes from the to the This is with previous results that Rlp24 Nog1 and from pre-60 S particles at cytoplasmic steps of biogenesis (13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google Scholar). of Nsa2 in the of S when was was from the S of but it could be in the whereas to S complexes. This of in was by We the on whole for pre-ribosomal factors Nog1, and Nog2, with used as a control In the absence of factors in the steps of the S subunit maturation from the yeast in the absence of the factor, Nog2, Nsa2 was still and to This was the of Nsa2 in in which various pre-60 S factors Nsa2 levels when upstream factors as Nog1 but when factors as Lsg1 of the we for Nsa2 in the and for 2 at 25 Nsa2 levels in from the at than at In the growing Nsa2 levels at As a was in Nsa2 levels in the the the results are of the of the that Nog1 was with in the that the the protein at We the of Nsa2 was of at the protein In a of the we in the levels of NSA2 when ribosome biogenesis was blocked This that the Nsa2 protein and its was these results that Nog1 is necessary for the of Nsa2 in pre-60 S particles, but when ribosome biogenesis is blocked upstream of Nsa2, this is in yeast ribosome biogenesis upstream of the necessary for adequate of Nsa2 to the and this pathway is Nsa2 is This Nsa2, when are Nog1 to pre-60 S particles 13Saveanu C. Namane A. Gleizes P.E. Lebreton A. Rousselle J.C. Noaillac-Depeyre J. Gas N. Jacquier A. Fromont-Racine M. Mol. Cell. Biol. 2003; 23: 4449-4460Crossref PubMed Scopus (171) Google and but it is still in in the Nsa2 a of Nsa2 when ribosome biogenesis was blocked to the of this protein of ribosomal biogenesis we the of Nsa2 after a with that of other pre-60 S we the in for various in which a pre-60 S was control of the a to the growing than from other pre-60 S factors Nog1, Nog2, the factor, This is with Nsa2 a short essential with other pre-60 S The of was for in a to and with that of in a We a of with that of This was to a in the at the and mRNAs disappeared when the to whereas the proteins could still be for The was in by proteins with The these which that the between Nog1 and Nsa2 was to the of the We that Nsa2 is than Nog1. A process could levels of this in in with the of ribosome biogenesis. Nsa2 an S this work, we Nsa2 as a pre-ribosomal required for adequate processing of the ITS2, which separates 25 S and 5.8 S rRNA. A number of pre-60 S factors, as Nog1 are necessary for this of maturation. The Nsa2 is that its in to on the of ribosome biogenesis. Indeed, in the biogenesis of the large ribosomal subunit was blocked upstream of Nsa2, this protein was in whole whereas expression was This is to a of pre-60 S they still be in Nsa2 is a for the of S biogenesis at a of the that Nsa2 play a part in the control of pre-60 S levels of Nsa2 the of pre-60 S particles through the these particles then birth to of large ribosomal in the cytoplasm. pre-60 S maturation is upstream of Nsa2, a could for a of Nsa2 be that the absence of Nsa2 could the pre-60 S particles from toward the cytoplasm. This could the absence of pre-60 S factors results in in the of these be an of the absence of upstream pre-60 S factors on Nsa2 cellular It is that Nsa2, as its human is in in to The of Nsa2 in with a short of this protein in with other pre-60 S date, we this is to the protein for this is that the of Nsa2 in in with the subunit of the A. P. N. P. J. L. D. J. S. O. M. EMBO 2001; PubMed Scopus Google Scholar). when pre-60 S biogenesis is Nsa2 be to the Nsa2 in the is one of the most conserved proteins between S. cerevisiae and F. E. S. R. B. N. R. G. G. 2001; PubMed Scopus Google Scholar). of this protein be found in eukaryotic with a of We that expression of the human orthologue of the TINP1 was able to in a in which Nsa2 was This that the sequence but the of the protein was conserved during its In Nsa2 to be tightly regulated. Indeed, TINP1 for as its production was in to with S. Zhang and D. I. as annotated in the Nsa2 in as a for as a for ribosome biogenesis. In to these expression TINP1 is located in the human in the which is in with X. G. M. M. B. D. E. G. S. 1999; PubMed Scopus Google Scholar). that together with other found in this a X. G. M. M. B. D. E. G. S. 1999; PubMed Scopus Google Scholar). that levels of are by and the of the M. M. R. M. H. J. 2004; PubMed Scopus Google Scholar). be that of TINP1 expression could participate in the of the of of by The here that expression of and is required for production of for A of this tightly of Nsa2 on S the work, we have that Nog1 to pre-60 S particles to we could in between the proteins in E. that Nog1 and Nsa2 this was it still that the proteins are to other on complexes The first of Nog1, which with Nsa2 in are conserved between yeast and eukaryotes than the part of the It that the a for the of the with we could the physical was the whole of pre-60 S particles to it a between Nog1 and Nsa2 a which could be pre-60 S complexes but when the proteins are in for the of Nsa2 on pre-60 S complexes was by In a with Nsa2 as we found ribosomal proteins as and C. and M. when at the of the in the of the mature large ribosomal subunit, proteins are on the on the of the that Nsa2 to the of the large subunit in the of this have characterized Nsa2 as a pre-60 S required for the processing of the 27 SB This physical and functional partner of Nog1 to be an factor, in with the of ribosome biogenesis. with its during its these Nsa2 and its for the of is to the of cellular We J. L. F. and F. for and and C. for with production of We are to F. B. and the in for the and the We for the yeast with Nsa2 as a The was from and was by

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