Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3′-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3′-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Diamond Blackfan anemia (DBA) 5The abbreviations used are: DBA, Diamond Blackfan anemia; FISH, fluorescence in situ hybridization; rRNA, ribosomal RNA; ITS1, internal transcribed spacer 1; TAP, tandem affinity purification; DAPI, 4′, 6-diamidino-2-phenylindole. is a severe hypoplastic anemia that generally presents early in infancy (1.Freedman M.H. Baillieres Clin. Hematol. 2000; 13: 391-406Crossref PubMed Scopus (23) Google Scholar, 2.Willig T.-N. Gazda H. Sieff C.A. Hematology. 2000; 7: 85-94Google Scholar). Other clinical features of DBA are heterogeneous with some patients presenting craniofacial abnormalities, growth failure, predisposition to cancer, and other congenital abnormalities. Most cases of DBA arise spontaneously, with only a small proportion exhibiting familial transmission typically showing autosomal dominant inheritance. Approximately 25% of the DBA cases have been linked to mutations in the gene encoding ribosomal protein S19 (RPS19), and in these cases haploinsufficiency for this ribosomal protein gives rise to the disease (3.Draptchinskaia N. Gustavsson P. Andersson B. Pettersson M. Willig T.N. Dianzani I. Ball S. Tchernia G. Klar J. Matsson H. Tentler D. Mohandas N. Carlsson B. Dahl N. Nat. Genet. 1999; 21: 169-175Crossref PubMed Scopus (668) Google Scholar, 4.Willig T.N. Draptchinskaia N. Dianzani I. Ball S. Niemeyer C. Ramenghi U. Orfali K. Gustavsson P. Garelli E. Brusco A. Tiemann C. Perignon J.L. Bouchier C. Cicchiello L. Mohandas N. Tchernia G. Blood. 1999; 94: 4294-4306PubMed Google Scholar). The remaining cases are of unknown etiology. The Rps19 protein is a component of the small ribosomal subunit, and as such, defects in some aspect of ribosome structure, function, or synthesis may be an underlying cause of DBA. The human Rps19 protein belongs to a family of ribosomal proteins restricted to eukaryotes and archea. Rps19 does not have a homolog in the eubacterial ribosome where the properties of individual ribosomal proteins have been most extensively studied. As such, little is known regarding the function of members of the eukaryotic Rps19 family. In prokaryotes, ribosomal proteins play critical roles in ribosomal assembly through their interactions with each other and rRNA (5.Nomura M. Hill W.E. Dahlberg A. Garrett R.A. Moore P.B. Schlessinger D. Warner J.R. The Ribosome: Structure, Function, and Evolution. American Society for Microbiology, Washington, D. C.1990: 3-55Google Scholar). These interactions promote both steps in rRNA processing important for subunit maturation and the formation of active sites in mature subunits necessary for ribosome function. The precise functions of the ribosomal proteins in the production of the subunits in eukaryotes are poorly characterized, which calls for a more detailed investigation of the role played by Rps19 in ribosome synthesis and function. The yeast Saccharomyces cerevisiae has proven to be an outstanding system to investigate factors involved in eukaryotic ribosome synthesis, including ribosomal proteins (6.Baudin-Baillieu A. Tollervey D. Cullin C. Lacroute F. Mol. Cell. Biol. 1997; 17: 5023-5032Crossref PubMed Scopus (59) Google Scholar, 7.Ford C.L. Randall-Whitis L. Ellis S.R. Cancer Res. 1999; 59: 704-710PubMed Google Scholar, 8.Tabb-Massey A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, 9.Jakovljevic J. de Mayolo P.A. Miles T.D. Nguyen T.M. Leger-Silvestre I. Gas N. Woolford J.L. Mol. Cell. 2004; 14: 331-342Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). To date, ribosome synthesis in yeast has been broken down to a number of well characterized intermediates, and numerous factors required for discrete steps in the pathway have been identified (11.Fatica A. Tollervey D. Curr. Opin. Cell Biol. 2002; 14: 313-318Crossref PubMed Scopus (425) Google Scholar, 12.Fromont-Racine M. Senger B. Saveanu C. Fasiolo F. Gene. 2003; 313: 17-42Crossref PubMed Scopus (480) Google Scholar, 13.Tschochner H. Hurt E. Trends Cell Biol. 2003; 13: 255-263Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Ribosomal RNAs are initially synthesized as a polycistronic transcript containing 18 S, 5.8 S, and 25 S rRNAs (14.Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (652) Google Scholar). The mature rRNAs are derived from the primary transcript by a series of processing steps most of which occur within the nucleolus. Pre-rRNA processing occurs concomitantly with the assembly of ribosomal proteins onto the rRNA transcripts. The earliest pre-rRNAs are found in a large ribonucleoproteic precursor of 90 S, which splits into pre-40 S and pre-60 S particles after endonucleolytic cleavage in the internal transcribed spacer 1 (ITS1) at site A2 (see Fig. 5). Through proteomic analysis, non-ribosomal factors specifically associated with these pre-ribosomal particles at various maturation stages have been identified (15.Milkereit P. Kuhn T. Gas N. Tschochner H. Nucleic Acids Res. 2003; 31: 799-804Crossref PubMed Scopus (47) Google Scholar). Pre-40 S particles have been found to contain a set of specific nonribosomal proteins that associate in the nucleus and accompany the particles into the cytoplasm where they are required for the final maturation of the small subunit (16.Schafer T. Strauss D. Petfalski E. Tollervey D. Hurt E. EMBO J. 2003; 22: 1370-1380Crossref PubMed Scopus (248) Google Scholar). In yeast, Rps19 is encoded by duplicated genes, and which from by a amino The yeast Rps19 proteins have with the human protein The the of the yeast and human proteins and of of these in is a for amino acid substitutions in patients with DBA T.N. Draptchinskaia N. Dianzani I. Ball S. Niemeyer C. Ramenghi U. Orfali K. Gustavsson P. Garelli E. Brusco A. Tiemann C. Perignon J.L. Bouchier C. Cicchiello L. Mohandas N. Tchernia G. Blood. 1999; 94: 4294-4306PubMed Google Scholar). Other amino acid substitutions found in DBA patients that occur of this in that are either or have substitutions yeast and this of on the function of the yeast Rps19 protein into the function of human homolog and in Diamond Blackfan Here we show that Rps19 is an essential protein that is required for the synthesis of the small ribosomal in cells is linked to a pre-rRNA processing at site A2 and to the of 40 S subunit which fail to non-ribosomal factors involved in subunit In a similar in pre-rRNA processing is by the of yeast Rps19 mutations found in DBA These results that Rps19 is a protein in ribosome and a or of function of the protein Rps19 and a in pre-rRNA processing and subunit and yeast used in this by the Saccharomyces deletion and from or used in yeast yeast and and yeast amino and to in by F. Google Scholar). In some as a by and on yeast and to in to 25% of that used in yeast with of and and growth by a of of the to the of of both Rps19 The gene from by and into I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). into a yeast heterozygous for and by and from and on with as initially for growth on to growth The of the of and in these the in each identified that growth on the of of the The used in this and of by The into mutations in for DBA patients introduced at in the gene the introduced into by which the of mutations the either or of into the growth on The functions of on the after growth to containing and on as by F. Mol. Cell. Biol. PubMed Scopus Google Scholar). generally at for in an and at an and a or from yeast by Nucleic Acids Res. PubMed Scopus Google on a and to a In Fig. the at with the 18 S and 25 S In Fig. at with and The with the in or in and by mutations found in DBA patients rRNA processing and the of to growth in The with a of the of growth in and with as to to and on containing or on the are each in and with as to and for cells and with and a to mature 18 S rRNA. each the of S pre-rRNA to 18 S rRNA derived from The in cells with set at Other the S rRNA with a to the of for in to the as by with the of pre-rRNA as C.L. Randall-Whitis L. Ellis S.R. Cancer Res. 1999; 59: 704-710PubMed Google Scholar). The used in the initially in containing and by of Rps19 by to containing in cells from 40 of each by from both each in 1 of and for at with of cells in into containing 1 and either on or for or at the cells on to the of on and to for at and to at a from to 1 The time from the that the in the deletion are of in the and after the to the of with the and of of and of RNAs as C. J. M. Mol. Cell. Biol. 2002; 22: PubMed Scopus Google Scholar). of proteins as I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). for at with in with and with with a on a the in and of pre-rRNA by as by Gleizes J. Leger-Silvestre I. F. D. Gas N. J. Cell Biol. PubMed Scopus Google Scholar). The of the is where to for by The to for in containing for by and to for in The for at and 1 at Rps19 for Cell the to which yeast growth and is on the genes, for or the other gene from the to a heterozygous for both the and the from individual for growth on at The results from this that of either gene growth fail to both genes are To Rps19 is required for a where the of and are and a of is of the These that growth cells are to and is that the yeast Rps19 proteins are essential for growth and of Rps19 the of 40 S either gene the we from and deletion and these through to a of ribosomal subunits and in protein Fig. that from the of 40 S subunits and to The of ribosome synthesis in the is which is to the 40 S subunit and is with growth with the that the of 40 S subunits and S subunits after a to with these the on ribosome synthesis are not as as those in the for the deletion are similar to results for of genes encoding other ribosomal proteins that are essential of the 40 S ribosomal subunit A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, M. Ellis S.R. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of 18 S rRNA in the of of 40 S subunits in deletion be the of a in subunit synthesis or subunit through the yeast pathway in and cells by The in containing for to Rps19 in the and ribosomal with a of The with which is used in the of ribosomal occurs early in the synthesis of rRNA, the primary after in the of Fig. that the cells the S to S and S which to 25 S and 18 S rRNAs in the first of the The in processing to the S and S the some in the production of 25 S rRNA of Rps19, the of 25 S rRNA by to of that observed in The production of 18 S rRNA, on the other severely in the of maturation of the 18 S rRNA after synthesis of a late which to S pre-rRNA (see through the rRNA processing pathway in cells to steps to the production of 18 S rRNA are more severely those to 25 S rRNA. Rps19 for Pre-rRNA at more the pre-rRNA processing by the of Rps19, we on RNAs from cells either on or for and to to down Rps19 of the yeast rRNA processing pathway is in Fig. the of 25 S rRNA down Rps19 the of 18 S rRNA The in 18 S rRNA is by an in S and S that cells of Rps19 are at rRNA at the and sites within and with an that to the and A2 cleavage sites a severe of the of S a in the pathway to 40 S and an in to be S which from the of mature 18 S rRNA to the cleavage site to the that this S pre-rRNA that the S rRNA the A2 cleavage cells of Rps19 are in cleavage at the A2 site within ITS1, as in Fig. The of S and S rRNA in the cells that the primary is in with on cleavage within with on the cleavage at sites and A2 sites (14.Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (652) Google Scholar). In the precursor in cells of Rps19 for the of Rps19 on cleavage at the A2 site In the of and only a in cells that cleavage of S and S at site in the pathway to mature 25 S rRNA. similar pre-rRNA only gene the processing observed in these accumulation of S pre-rRNA not These that Rps19 is necessary for maturation of the pre-rRNA and the of this ribosomal protein for processing at the A2 site in the of Pre-40 S the of the S particles of Rps19 and they be to the cytoplasm or are retained in the nucleus and Fig. the by fluorescence in situ of the pre-40 S particles with a to the of ITS1, in and As observed I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google cells in the where most ribosome is and in the cytoplasm where of the S pre-rRNA to 18 S mature In cells of Rps19, a of the which that the pre-40 S particles are from the to the small subunit in the nucleus with most of the restricted to the which be from the to with with a to the a in cells and not in with the accumulation of pre-40 S particles containing S pre-rRNA not deletion of or is to induce a similar These are of of cells of which is required for cleavage at the A2 cleavage site within A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, 10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). The is with a in early pre-rRNA cleavage steps in the nucleolus. with the accumulation of pre-rRNA in the in a of cells is the that the with the and to from the this at a by In we observed to be the the nucleolus. These strongly that of the nucleus is by of Rps19, a to be involved in of ribosome P. S. E. E. M. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google Scholar). Rps19 for of into the at which the assembly of the 40 S is of Rps19, we of non-ribosomal factors with pre-rRNAs in the used genes encoding Enp1, Tsr1, and as a of the of these proteins with pre-ribosomal In is found associated with 90 S and are in pre-40 S is found associated with both of The proteins with and the pre-rRNAs by with the of the 25 S rRNA, which does not associate with these that of this to In with found in the 90 S particles S and not with S As the precursor containing S rRNA in in with Enp1, Tsr1, and Rio2. of Rps19, this The of pre-rRNA with Enp1, Tsr1, and that these proteins associate with in the of Rps19. the of S pre-rRNA with these which that some of the proteins associated with the 90 S in the pre-40 S which in the of Rps19. the proteins by As in Fig. which is a protein in in to the in similar to that of the pre-40 S particles with the of with the S pre-rRNA by In the late as in and from in cells to in The of these two proteins within the nucleus and from the which these not to pre-40 S these proteins are in the These show that Rps19 is necessary for of non-ribosomal factors found associated with pre-40 S of these factors may processing at site A2 and maturation of the to the 18 S rRNA. in DBA and rRNA When into the The results that of and rRNA processing by with cleavage at the A2 site within Most of the mutations identified to in the gene of DBA patients be similar to these either through large or mutations that with the production of Rps19 protein. In patients have mutations in the which are for the disease a identified in a DBA which results in a for at in the human protein. occurs in the Rps19 to the for mutations in DBA has not been in the of and as such, may a Cancer this or other mutations associated with DBA Rps19 function has not been of these mutations in of Rps19 yeast and we the of of these mutations on growth and pre-rRNA processing in The with a of of When this is on the of are the primary of Rps19 protein to As in Fig. the similar to the found in the DBA the of the protein to growth on that this has a severe effect on Rps19 function. other mutations in the human and have on the of the yeast protein to In two other in and in not the of the yeast protein to is a in DBA and as such, not be to have an on Rps19 function. the of these mutations on ribosome synthesis by from each of these after from to that where growth is by the of a in show S rRNA to where growth is not the pre-40 S particles in these retained in the nucleus and in the as by in situ is a the of mutations to growth in yeast and their to the function of Rps19 in ribosome biogenesis. The gene encoding ribosomal protein S19 is the only gene known to be involved in the of Diamond Blackfan Approximately 25% of DBA patients have mutations in The remaining cases of DBA are of unknown etiology. in the yeast S. cerevisiae have that some ribosomal proteins are required for the maturation of ribosomal subunits (6.Baudin-Baillieu A. Tollervey D. Cullin C. Lacroute F. Mol. Cell. Biol. 1997; 17: 5023-5032Crossref PubMed Scopus (59) Google Scholar, 7.Ford C.L. Randall-Whitis L. Ellis S.R. Cancer Res. 1999; 59: 704-710PubMed Google Scholar, 8.Tabb-Massey A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, 10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). These have linked individual ribosomal proteins to specific steps in rRNA processing subunit to yeast to a of the function of the Rps19 family of of either of the yeast genes a in growth and a of 40 S ribosomal subunits. of both genes is is with showing that a deletion in is to the that the Rps19 protein a critical role in growth and H. Draptchinskaia N. I. A. P. E. S. Dahl N. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). a critical role of Rps19 in the production of the small ribosomal The most by of Rps19 is a of cleavage at site A2 within and of the maturation of the 3′-end of the 18 S rRNA. The endonucleolytic cleavage at A2 within the and results in of pre-40 S and pre-60 S particles from the 90 S ribosomal of Rps19 affects the production of 40 S ribosomal as cleavage at site rise to a in the pathway to S subunits. The S pre-rRNA is not a of the 40 S production and may be into mature 18 S rRNA, as with some mutations in the gene C. S. 2000; PubMed Scopus Google Scholar, Nucleic Acids Res. 2004; PubMed Scopus Google Scholar). the S pre-40 S which in cells are retained in the nucleus and are at to mature 40 S subunits. In to the of the numerous other non-ribosomal factors with the for various steps in pre-rRNA processing and show that the aberrant pre-40 S particles that in cells the non-ribosomal factors Enp1, Tsr1, and which a in the assembly of the pre-40 S In and are found associated with pre-40 S subunits after the 90 S is by cleavage at are not required for A2 are for cleavage at site which mature 18 S rRNA in the cytoplasm I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar, E. Gleizes M. Mol. Cell. Biol. 2003; 23: PubMed Scopus Google Scholar). The of aberrant pre-40 S subunits found in cells of Rps19 to be a of the of these particles within the as is Tsr1, on the other is in the nucleus and the that the aberrant pre-40 S subunits may be to within the nucleolus. In is a component of early 90 S and late 40 S (16.Schafer T. Strauss D. Petfalski E. Tollervey D. Hurt E. EMBO J. 2003; 22: 1370-1380Crossref PubMed Scopus (248) Google Scholar). of show a accumulation of S rRNA at similar to the observed for J. A. Nucleic Acids Res. 2003; 31: PubMed Scopus Google Scholar). Rps19 and within the 90 S to cleavage at In the of a component of the small subunit in the aberrant pre-40 S particles that in cells that of factors specific to the 90 S particles may be these that Rps19 as an essential protein for some of the early steps in the maturation of to 40 S ribosomal subunits. Rps19 haploinsufficiency is to rise to DBA. In DBA patients mutations in an gene that the gene with or affects by or T.N. Draptchinskaia N. Dianzani I. Ball S. Niemeyer C. Ramenghi U. Orfali K. Gustavsson P. Garelli E. Brusco A. Tiemann C. Perignon J.L. Bouchier C. Cicchiello L. Mohandas N. Tchernia G. Blood. 1999; 94: 4294-4306PubMed Google Scholar, L. E. J.L. A. J.M. A. E. Niemeyer D. Sieff C.A. J. 2004; PubMed Scopus Google Scholar). DBA patients have been identified that have mutations in These those that arise spontaneously, are they are not found in the in family As such, they are as on the of The that of these mutations in of the human and yeast genes, to the effect of these amino acid substitutions on the function of the yeast protein. of the mutations including a have on Rps19 function in These mutations of pre-rRNA processing to those observed in cells Rps19, that they result in the of function of the protein. the two mutations that not the function of the yeast the is as such, not be to function. The the does not the function of the yeast protein is is that the to at in the human may be an a the of this on function may be more specific to the human protein and not be by the yeast protein. the of mutations in is important for in DBA, these a critical for the to Rps19 function in Other have been identified that result from defects in genes known to ribosome The of is by mutations in the which a involved in ribosomal processing and D. S. F. E. F. C. 2003; PubMed Scopus Google Scholar, J. S. M. U. S. A. 2004; PubMed Scopus Google the gene is involved in ribosomal D. J. U. S. A. 2004; PubMed Scopus Google Scholar). of these including DBA, has that from clinical with some in These that or factors the and of each Other that may result from defects in ribosome synthesis that show clinical M. H. K. C. B. G. S. I. de A. Cell. Full Text Full Text PDF PubMed Scopus Google and J. A. M. S. P. V. J. T. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. Ellis L. J.M. Nat. Genet. 2003; 33: PubMed Scopus Google Scholar). the to of ribosomal subunits is a to the clinical of a number of human and for their to this