Ssd1p of Saccharomyces cerevisiae Associates with RNA

The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4,slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm. The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4,slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm. Cellular RNAs do not exist as a free form but as an RNA-protein complex. The proteins that directly associate with RNA are thought to play important roles in the regulation of gene expression at the post-transcriptional level (1Atwater J.A. Wisdom R. VermaI I.M. Annu. Rev. Genet. 1990; 24: 519-541Crossref PubMed Scopus (212) Google Scholar, 2Bandziulis R.J. Swanson M.S. Dreyfuss G. Genes 3: 431-437Crossref PubMed Scopus (490) Google Scholar). In eukaryotic cells, proteins that bind to RNA polymerase II transcripts include both heterogeneous nuclear RNA-binding proteins and cytoplasmic mRNA-binding proteins. Heterogeneous nuclear RNA-binding proteins bind pre-mRNAs and are associated with them during the processing events required for the formation of mature mRNA (1Atwater J.A. Wisdom R. VermaI I.M. Annu. Rev. Genet. 1990; 24: 519-541Crossref PubMed Scopus (212) Google Scholar). Once mRNAs are transported to the cytoplasm, they form cytoplasmic mRNA-binding protein complexes (2Bandziulis R.J. Swanson M.S. Dreyfuss G. Genes 3: 431-437Crossref PubMed Scopus (490) Google Scholar). Cytoplasmic mRNA-binding proteins seem to regulate translation, localization, or stability of mRNA (3Hargrove J.L. Hulsey M.G. Beale E.G. BioEssays. 1991; 13: 667-674Crossref PubMed Scopus (73) Google Scholar). At present, many RNA-binding proteins have been isolated and characterized (4Sachs A.B. Cell. 1993; 74: 413-421Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 5Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1734) Google Scholar), but their functions have not been fully understood. In Saccharomyces cerevisiae, the SSD1 gene has been first characterized to suppress the sit4 mutation defective in a protein phosphatase subunit (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar). Not only in this case, but also in many other cases, SSD1 has been isolated as a single copy suppressor of mutation defective in RPC31encoding a subunit of RNA polymerase III (7Stettler S. Chiannilkulchai N. Denmat S.H. Lalo D. Lacroute F. Sentenac A. Thuriaux P. Mol. 239: 169-176Crossref PubMed Scopus (80) Google Scholar), in PDE2encoding the cyclic AMP phosphodiesterase (8Wilson R.B. Brenner A.A. White T.B. Engler M.J. Gaughran J.P. Tatchell K. Mol. Cell. Biol. 1991; 11: 3369-3373Crossref PubMed Scopus (50) Google Scholar), in BCK1encoding mitogen-activated protein kinase kinase kinase (9Costigan C. Gehrung S. Snyder M. Mol. Cell. Biol. 1992; 12: 1162-1178Crossref PubMed Scopus (202) Google Scholar), inMPK1 encoding mitogen-activated protein kinase (10Lee K.S. Irie K. Gotoh Y. Watanabe Y. Araki H. Nishida E. Matsumoto K. Levin D.E. Mol. Cell. Biol. 1993; 13: 3067-3075Crossref PubMed Scopus (312) Google Scholar), or in G1 cyclin (11Cvrckova F. Nasmyth K. EMBO J. 1993; 12: 5277-5286Crossref PubMed Scopus (141) Google Scholar). These reports indicate that SSD1is involved in many systems. Sutton et al. also reported that there are two alleles of the SSD1 gene; one is calledssd1-d (dead) and the other is called SSD1-V(viable). They described that SSD1-V could suppress the double mutations of ssd1-d and sit4 (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar). We have also isolated the SSD1 gene as the MCS1 gene involved in stable maintenance of the minichromosome (12Uesono Y. Fujita A. Toh-e A. Kikuchi Y. Gene ( Amst. ). 1994; 143: 135-138Crossref PubMed Scopus (42) Google Scholar). TheSSD1/MCS1 gene product was detected as a ∼160-kDa protein in certain wild type strains bearing SSD1-V, such as KA31 or RAY-3A, whereas a protein of this size was not detected in another wild type strain bearing ssd1-d, such as YPH499 (7Stettler S. Chiannilkulchai N. Denmat S.H. Lalo D. Lacroute F. Sentenac A. Thuriaux P. Mol. 239: 169-176Crossref PubMed Scopus (80) Google Scholar). These findings indicate that SSD1-V is simply a wild type gene andssd1-d is a defective gene. However, the functions ofSSD1 have not yet been clarified. In recent years, it has been reported that SSD1 has a weak but significant similarity with dis3 + ofSchizosaccharomyces pombe (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar, 13Kinoshita N. Goebl M. Yanagida M. Mol. Cell. Biol. 1991; 11: 5839-5847Crossref PubMed Scopus (68) Google Scholar), DSS1 ofS. cerevisiae (14Dmochowska A. Golik P. Stepien P.P. Curr. Genet. 1995; 28: 108-112Crossref PubMed Scopus (56) Google Scholar), vacB of Shigella flexneri (15Tobe T. Sasakawa C. Okada N. Honma Y. Yoshikawa M. J. Bacteriol. 1992; 174: 6359-6367Crossref PubMed Google Scholar), cyt4 of Neurospora crassa(16Turcq B. Dobinson K.F. Serizawa N. Lambowitz A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1676-1680Crossref PubMed Scopus (20) Google Scholar), zam of Synechocytosis PCC 6803 (17Beuf L. Bedu S. Cami B. Joset F. Plant Mol. Biol. 1995; 27: 779-788Crossref PubMed Scopus (5) Google Scholar), andrnb of Escherichia coli (18Zilhao R. Camelo L. Arraiano C.M. Mol. Microbiol. 1993; 8: 43-51Crossref PubMed Scopus (38) Google Scholar). Some of these genes are known, or implied, to be involved in the modification of RNAs: 1)cyt4 is required for the mitochondrial rRNA splicing and processing reaction; 2) DSS1 is a multicopy suppressor of the disruptant of SUV3 encoding a putative RNA helicase-like protein; 3) the vacB mutation reduces the level of the virulence antigens, IpaB, IpaC, IpaD, and VirG, at the post-transcriptional level; and 4) the RNase II encoded byrnb has a 3′-to-5′ exoribonuclease activity. However, there have been no reports describing direct interaction with RNA in these gene products, except for RNase II of E. coli. Here we report the biochemical characterization and cellular localization of the Ssd1 protein. RAY-3A (MATa ura3 leu2 trp1 his3) and YRM1H (RAY-3A ssd1Δ::HIS3) were used for the ribonuclease assay, nucleotide binding studies, metabolic labeling with 32Porthophosphate, and sucrose gradient fractionation of cell extract. KA31–2A (MATa ura3 leu2 trp1 his3) (19Irie K. Takase M. Lee K.S. Levin D.E. Araki H. Matsumoto K. Oshima Y. Mol. Cell. Biol. 1993; 13: 3076-3083Crossref PubMed Scopus (259) Google Scholar) and YKM1H (KA31–2Assd1Δ::HIS3) were used for testing growth rates and for indirect immunofluorescence microscopy. The SSD1disruption was performed by using pYK907 plasmid as described previously (12Uesono Y. Fujita A. Toh-e A. Kikuchi Y. Gene ( Amst. ). 1994; 143: 135-138Crossref PubMed Scopus (42) Google Scholar). Culture media, including YPD (1% yeast extract, 2% peptone, and 2% glucose) and synthetic minimal SD (0.7% yeast nitrogen base without amino acid and 2% glucose) with amino acid supplements, were prepared according to Rose et al. (20Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: Alaboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). SRaf contained 0.7% Difco yeast nitrogen base without amino acids, 2% raffinose, and appropriate supplements. Plasmid pFK4 was constructed by inserting the 6-kilobase BamHI fragment bearing theSSD1 gene derived from the original clone, pFK2CU (12Uesono Y. Fujita A. Toh-e A. Kikuchi Y. Gene ( Amst. ). 1994; 143: 135-138Crossref PubMed Scopus (42) Google Scholar), into pUC13. For overexpression of the SSD1 gene, a plasmid pSSD1.0 was made as follows. pYES2 (Invitrogen), a high copy number plasmid carrying the URA3 marker and the inducibleGAL1 promotor, was digested with HindIII, and the DNA ends were made flush with a Klenow fragment of DNA polymerase I, digested with BamHI, and then ligated with the 5.2-kilobaseHpaI-BamHI fragment bearing the SSD1gene derived from pFK4. Translational initiation of Ssd1.0p is started at the ATG at position 52 in the open reading frame of SSD1. Therefore, Ssd1.0p expressed from pSSD1.0 plasmid encodes the protein lacking 17 amino acids of the N terminus. pSSD1.1 was constructed as follows. After pSSD1.0 was digested with XhoI andHindIII, the DNA ends were made flush and ligated. pSSD1.2 was constructed by digestion of pSSD1.0 with XbaI, and the resulting large fragment was recircularized. Plasmid pFK1CU or plasmid pFK5EU contains SSD1 on YCUp4 or YEUp3 vector (constructed by Fujita), respectively (12Uesono Y. Fujita A. Toh-e A. Kikuchi Y. Gene ( Amst. ). 1994; 143: 135-138Crossref PubMed Scopus (42) Google Scholar). YRM1H carrying or pSSD1.2 were in to and to by at for For the of cellular extract, were by and with and of and were in of and by with an of for of was and were for was and at for to cell of Ssd1p and were performed as described previously (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar). RNA binding was by the proteins with RNA as described previously J. J. Biol. 1993; Full Text PDF PubMed Google Scholar). RNA were prepared from yeast by using RNA and with Yeast of YRM1H carrying pFK1CU or YCUp4 were in to at of YRM1H carrying pSSD1.0 in were to or 2% and for at Cellular were prepared from as described and were performed using of anti-Ssd1p as described previously (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar). were with for of exoribonuclease and in of the and exoribonuclease were The of exoribonuclease were on the of from or RNA according to the as described previously E. N. Y. T. M. N. Yanagida M. U. S. T. EMBO J. PubMed Scopus Google Scholar, J. R. Arraiano C.M. Microbiol. 1995; PubMed Google Scholar, A. J. Biol. Full Text PDF PubMed Google Scholar). For of yeast RAY-3A were in YPD to and of YRM1H carrying or pSSD1.2 were in for at were by with in of binding and of and an of binding was and cellular were prepared by as described this extract, single-stranded DNA DNA and binding assay, were performed as described previously M.J. Dreyfuss G. Mol. Cell. Biol. 1993; 13: PubMed Scopus (38) Google Scholar). Indirect immunofluorescence of yeast was performed with a modification of previously T. Methods 1989; Scholar). KA31–2A carrying a multicopy plasmid carrying the SSD1 gene, were to in and was directly to a of After for at were with and in of of and and for at were with and with were in of the of anti-Ssd1p and for at were to for and with were with the antibody for at were with and with For the of extract, were by with in of and of and were by for at one of the was with RNase at for and the other was the without S. M. Y. Y. A.M. Mol. Cell. Biol. PubMed Scopus Google Scholar). was of a sucrose gradient and at in a for at For a were with and were as described previously A.B. Cell. 1989; Full Text PDF PubMed Scopus Google Scholar). For the of that contains were and cell were prepared by E. J. J. Bacteriol. PubMed Google Scholar). were a sucrose gradient by at in a for at of the gradient was using the and has been reported that the Ssd1 protein weak similarity with the which is to a mitochondrial RNA splicing and processing of N. B. Dobinson K.F. Serizawa N. Lambowitz A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1676-1680Crossref PubMed Scopus (20) Google Scholar). report demonstrated the conserved domains in proteins not only but also RNase II, a to encoded by of E. coli (14Dmochowska A. Golik P. Stepien P.P. Curr. Genet. 1995; 28: 108-112Crossref PubMed Scopus (56) Google Scholar). Therefore, we the amino acid of Ssd1p with of other proteins. analysis using the revealed that Ssd1p has with of S. pombe in amino acids of S. cerevisiae in amino E. N. Y. T. M. N. Yanagida M. U. S. T. EMBO J. PubMed Scopus Google Scholar), of S. cerevisiae in amino of N. in amino of Shigella in amino and of E. in amino The homology demonstrated that are to the of Ssd1p and that conserved domains exist in this The first domain of Ssd1p was a from to the was a from to and the was a from to in these domain is the most conserved among these proteins In to the proteins in the protein from the of also has these These findings indicate that domains and are conserved from to these conserved domains are necessary for the of SSD1 or we constructed SSD1 encoding a protein lacking 17 amino acids of the N of Ssd1p expressed on a multicopy vector and could the of disruptant of the KA31–2A it was expressed from the on SRaf that encoded a protein In to the gene lacking both domains and or of domains and could not the of YKM1H overexpression in the of the growth of YKM1H at or not the SSD1 gene the including domains and for In the of dis3 + of S. a that the most conserved is necessary for has been described previously N. Goebl M. Yanagida M. Mol. Cell. Biol. 1991; 11: 5839-5847Crossref PubMed Scopus (68) Google Scholar). The not growth was in the strain KA31 or in another wild type RAY-3A not the of Ssd1p has similarity with that of the RNase II of E. coli as described we Ssd1p had an exoribonuclease activity. The were prepared by using anti-Ssd1p antibody from the of the wild the and these we an ability of Ssd1p to or RNA from yeast the of RNase II coli J. R. Arraiano C.M. Microbiol. 1995; PubMed Google Scholar), to exoribonuclease of S. cerevisiae A. J. Biol. Full Text PDF PubMed Google Scholar), or mitochondrial to exoribonuclease ofS. cerevisiae J. J. Biol. 1993; Full Text PDF PubMed Google Scholar). However, we were to exoribonuclease with these We Ssd1p could bind RNA in Ssd1.0p and from it were from of yeast SSD1 using anti-Ssd1p antibody and a The of and were and detected by analysis The of and from amino acid were and suggesting that of these proteins are and that the in the of which not significant similarity with other proteins. The was RNA from yeast of lacking both and could bind RNA, Ssd1.0p and could not that associate with RNA directly without binding proteins and that the is necessary for RNA The Ssd1.0p not bind to RNA is at this We a that Ssd1.0p and bind it is important to the Ssd1p has an ability to bind RNA. an ability of Ssd1p to bind we The of from the yeast Ssd1.0p or was with and in or and proteins were with Ssd1.0p was to bind to at but not to whereas lacking conserved domains bound but not Ssd1.0p has an ability to bind single-stranded and the conserved seem to be necessary for However, lacking the most conserved domain could bind Ssd1.0p and These indicate that Ssd1p bind without the most conserved as in the of and that a other the conserved bind Interestingly, could bind as well as suggesting that the including both domains and is necessary for binding to single-stranded the RNA binding of we Ssd1p or was bound to binding has been in the of a of RNA-binding proteins M.J. Dreyfuss G. Mol. 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Biol. 1993; 13: PubMed Scopus (38) Google Scholar). from the wild type were with and in or The proteins were with Ssd1p bound to the of in whereas it bound to to the other a high The was using the of Ssd1.0p lacking both domains and to bind with in that the binding of Ssd1p on the including both domains and is important to the localization of Ssd1p the proteins their functions in their Indirect immunofluorescence using anti-Ssd1p antibody was to the localization of of the the SSD1 gene was on a multicopy YEUp3 vector to in The anti-Ssd1p antibody revealed an in and a weak in the in the disruptant carrying The was in the cytoplasm, of or that Ssd1p mainly in the the cell a were not in the disruptant carrying YEUp3 vector These that Ssd1p associate with RNA in the but not DNA or RNA in the Ssd1p play roles in the stability or of cytoplasmic RNA Ssd1p associate with cellular RNA or prepared from the wild type were on a sucrose and was by using anti-Ssd1p Ssd1p a in However, of with RNase and Ssd1p was mainly in the and the Ssd1 protein was to The that Ssd1p in the with also in the that of Ssd1p form complexes with certain protein or we that Ssd1p associated with RNA in Cytoplasmic mRNAs exist in translationally active form or translationally In cells, the mRNA at the and to be by a to In al. 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Biol. 1991; 11: 5839-5847Crossref PubMed Scopus (68) Google Scholar). Ssd1p also another for an exoribonuclease activity. in most of Ssd1p was in with and a of Ssd1p as stable Ssd1p form a stable not only with proteins but also with certain RNA and formation of such a be necessary for exoribonuclease activity. modification of Ssd1p also to the expression of exoribonuclease Ssd1p is in (12Uesono Y. Fujita A. Toh-e A. Kikuchi Y. Gene ( Amst. ). 1994; 143: 135-138Crossref PubMed Scopus (42) Google Scholar), suggesting that the yet ribonuclease of Ssd1p be by a certain protein lacking the conserved domain could bind RNA Ssd1.0p as in domain to the of Ssd1p with RNA. Ssd1p is domain be necessary for exoribonuclease RNA the SSD1 gene domain for as in The RNA-binding C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1734) Google Scholar) are not in the of Interestingly, the necessary for interaction with RNA to be a domain showing a weak similarity but not a conserved domain Therefore, the domain of Ssd1p be a RNA binding The DSS1 gene has been isolated as a multicopy suppressor of the disruptant of encoding putative RNA involved in mitochondrial RNA has been reported that the amino of is to have a mitochondrial and that the disruptant is but not in a (14Dmochowska A. Golik P. Stepien P.P. Curr. Genet. 1995; 28: 108-112Crossref PubMed Scopus (56) Google Scholar). these to in the other the of S. cerevisiae a high with the of S. pombe the E. N. Y. T. M. N. Yanagida M. U. S. T. EMBO J. PubMed Scopus Google Scholar), Ssd1p and In et al. E. N. Y. T. M. N. Yanagida M. U. S. T. EMBO J. PubMed Scopus Google Scholar) have also reported that was to the of S. et al. N. Goebl M. Yanagida M. Mol. Cell. 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However, rRNA prepared from the not in or in with rRNA prepared from wild type not In Ssd1p was not cofractionated with or with as in the of Ssd1p not seem to be We could not Ssd1p in not Therefore, we have no to indicate direct interaction of Ssd1p with analysis using anti-Ssd1p antibody revealed that the protein in used as wild type strain was detected as an protein not protein be a protein lacking of the domain and suggesting that the protein also be a protein. The protein of another wild type was also the size as that of not These that a ssd1-d mutation is among many The in a such as (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar) and However, it is with another mutation such as the double mutation a of the single mutations (6Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar, S. Chiannilkulchai N. Denmat S.H. Lalo D. Lacroute F. Sentenac A. Thuriaux P. 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EMBO J. 1990; PubMed Scopus Google Scholar), 3) Y. Irie K. Matsumoto K. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar), and 4) rpc31, (7Stettler S. Chiannilkulchai N. Denmat S.H. Lalo D. Lacroute F. Sentenac A. Thuriaux P. Mol. 239: 169-176Crossref PubMed Scopus (80) Google Scholar). These by in to the that Ssd1p is a cytoplasmic RNA-binding that SSD1 be involved in the expression of genes at the post-transcriptional level by RNA