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

Abstract

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 underexpressing 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 anemiaassociated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Diamond Blackfan anemia (DBA)5 is a severe hypoplastic anemia that generally presents early in infancy (1, 2). 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, 4). 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). 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...

RPS19 mutations in patients with Diamond-Blackfan anemia

“…This is consistent with the finding that RPS19 is essential for the maturation of 18S rRNA [Flygare et al, 2007;Choesmel et al, 2007;Idol et al, 2007] and assembly of pre-40S particles [Léger-Silvestre et al, 2005]. Depletion of Rps19 in yeast causes strong inhibition of pre-rRNA cleavage at site A2 within ITS1 and of the subsequent maturation of the 3 0 end of 18S rRNA [Léger-Silvestre et al, 2005]. Similar effects are observed in human cells [Flygare et al, 2007;Choesmel et al, 2007;Idol et al, 2007].…”

“…The entire RPS19 interactome has been published: interestingly, most interactors are nucleolar, and include many RPs, preribosome components, and helicases [Orrù et al, 2007]. This is consistent with the finding that RPS19 is essential for the maturation of 18S rRNA [Flygare et al, 2007;Choesmel et al, 2007;Idol et al, 2007] and assembly of pre-40S particles [Léger-Silvestre et al, 2005]. Depletion of Rps19 in yeast causes strong inhibition of pre-rRNA cleavage at site A2 within ITS1 and of the subsequent maturation of the 3 0 end of 18S rRNA [Léger-Silvestre et al, 2005].…”

“…eADA activity is thus a useful test for identification of DBA associated with an RPS19 mutation. Some RPS19 mutations, i.e., p.Ala61Glu, p.Gly120Ser, p.Arg62Gln, and p.Arg62Trp, have little impact on cell growth and on 21S pre-rRNA accumulation in yeast [Léger-Silvestre et al, 2005;Gregory et al, 2007], whereas patients who carry them present classic DBA phenotypes, including malformations, high eADA levels, and variable response to steroids. p.Arg62Trp, in particular, is usually associated with a poor response to steroids and transfusion-dependence [Campagnoli et al, 2004].…”

Nucleus-Vacuole Junctions and Piecemeal Microautophagy of the Nucleus inS. cerevisiae

“…4 Studies of the yeast homolog of human Diamond Blackfan Anemia-associated protein, Rps19p, are consistent with this type of role. 64 Rps19p functions in ribosome biogenesis and is required for the maturation of 18S ribosomal RNA. 64 Depletion of Rps19p leads to an accumulation of aberrant preribosomal material in the nucleolus and the apparent upregulation of PMN.…”

Anémie de Diamond-Blackfan

“…Ce processus se déroule principalement dans le nucléole, un domaine spécialisé du noyau [21]. Trois des 4 ARN Chez S. cerevisiae, l'interruption de la synthèse de RPS19 entraîne un clivage anormal dans l'ITS1 [23]. Plusieurs facteurs pré-ribosomiques spécifiquement associés à la particule pré-40S ne sont plus incorporés.…”