Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.The synthesis of ribosomes is one of the major metabolic pathways of a cell. In Saccharomyces cerevisiae, ribosome assembly begins in the nucleolus after the transcription of two rRNA precursors, the 35S RNA (precursor of the 18S, 5.8S, and 25S rRNAs) and the pre-5S RNA, by RNA polymerases I and III, respectively. The synthesized pre-rRNAs are modified extensively at multiple positions specified by small nucleolar ribonucleoparticles (snoRNPs) or specific enzymes (1,22,33). During rRNA maturation, the 5Ј and 3Ј external transcribed sequences (ETS) and internal transcribed sequence 1 (ITS1) and ITS2 are removed from the 35S precursor RNA by wellordered cleavages and trimming events, which require the enzymatic activities of helicases and endo-and exonucleases (19,37).Cotranscriptional assembly of ribosomal and nonribosomal proteins in the nucleolus gives rise to a large ribonucleoprotein particle corresponding to the 90S preribosomal complexes described more than 20 years ago (35) and recently characterized biochemically (8,14). These early preribosomal complexes are further converted to smaller pre-40S (43S) and pre-60S (66S) particles, precursors of the mature small and large ribosomal subunits. The pre-40S complexes, each containing a precursor of the 18S rRNA, are exported into the cytoplasm, where they give rise to the mature 40S ribosomal particles (36). Most of the large ribosomal subunit proteins are absent from the 90S preribosomes (8,14) and associate in the nucleolus with the pre-rRNA, probably concomitantly with the formation of the pre-60S particles. During pre-60S particle maturation, 27S prerRNA intermediates are converted into 25S and 5.8S mature rRNAs by successive and well-ordered steps. Several pre-60S particles, which differ in their RNA and protein compositions,...
The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinitypurified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively.
The gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBArelated genes may be involved in the synthesis of the ribosomal subunits. IntroductionDiamond-Blackfan anemia (DBA) is a rare pure red blood cell aplasia of childhood characterized by the absence or decreased numbers of erythroid precursors in the bone marrow but an otherwise normal cellularity. Approximately 40% of the DBA patients present various somatic malformations that mostly occur in the cephalic area but also in the hand and/or limb, urogenital tract, and heart. [1][2][3] Clinical expression in DBA is highly heterogeneous, and evolution of the disease is unpredictable. Treatment includes steroid therapy and transfusion with iron chelation. Bone marrow or cord blood transplantation is the only curative treatment but requires an HLA-matched sibling and is mostly reserved to patients with severe complications.It has been established that 25% of the DBA patients bear a mutated allele of the gene encoding the ribosomal protein S19 (RPS19). [4][5][6] RPS19 is one of the 32 proteins that assemble with the 18S ribosomal RNA (rRNA) to form the small (40S) ribosomal subunit. RPS19 is an essential protein, as homozygous deletion of RPS19 in the mouse leads to embryonic lethality before implantation at the blastocyst stage. 7 A wide range of mutations have been identified in DBA patients, from missense to nonsense mutations and from partial to complete deletion of one allele. 3,5,6 Some missense mutations affect both the stability and the intracellular transport of RPS19. 8 Consistent with a role in DBA pathogenesis, depletion of RPS19 with specific siRNAs severely alters proliferation and differentiation of erythroleukemic cell lines or CD34 ϩ cells in culture. [9][10][11] Although DBA is to date the only genetic disease linked to mutation of an autosomal ribosomal protein gene, a number of other bone marrow failure symptoms (dyskeratosis congenita, cartilagehair hypoplasia, and Shwachman-Diamond ...
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