Impaired ribosome biogenesis in Diamond-Blackfan anemia

Abstract

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 ...

Ribosomopathies: human disorders of ribosome dysfunction

“…[22][23][24][25] Moreover, there is evidence that depletion of any of the RPS proteins causes a reduction in the amount of free 40S subunits and a significant reduction in the amount of mature 80S ribosomes (with the exception of RPS25, which has not been shown to be mutated in DBA). 4 In contrast, when RPL proteins, including RPL35A, are depleted, the amount of the 60S subunit is reduced, as is the level of mature 80S ribosomes.…”

Anémie de Diamond-Blackfan

“…Les particules pré-40S anormales formées en l'absence de RPS19 ne quittent pas le noyau et sont dégradées rapidement, et l'ARN 18S n'est pas produit. Un défaut de maturation similaire est observé après l'interruption de la synthèse de RPS19 grâce à des siARN dans des cellules humaines en culture [24][25][26]. Dans ce cas, le pré-ARN ribosomique 21S n'est plus clivé en ARN 18S-E, le dernier précurseur de l'ARN 18S.…”

“…En accord avec les données obtenues in vitro, des cellules de patients atteints d'ADB présentent un défaut de maturation du pré-ARNr reproduisant l'effet des siRNA contre la protéine ribosomique considérée [16,[24][25][26][27]. Ainsi, on retrouve un défaut de conversion de l'ARN pré-ribosomique 21S en ARN 18S-E dans des cellules médul-laires CD34 + (cellules souches et progéniteurs hématopoïétiques), ou dans des fibroblastes cutanés de patients porteurs de mutations dans RPS19 [24][25][26].…”

“…Ainsi, on retrouve un défaut de conversion de l'ARN pré-ribosomique 21S en ARN 18S-E dans des cellules médul-laires CD34 + (cellules souches et progéniteurs hématopoïétiques), ou dans des fibroblastes cutanés de patients porteurs de mutations dans RPS19 [24][25][26]. Un défaut similaire peut être observé dans des lignées lymphoblastoïdes établies à partir des lymphocytes de ces patients [24,26,27].…”

“…Ainsi, on retrouve un défaut de conversion de l'ARN pré-ribosomique 21S en ARN 18S-E dans des cellules médul-laires CD34 + (cellules souches et progéniteurs hématopoïétiques), ou dans des fibroblastes cutanés de patients porteurs de mutations dans RPS19 [24][25][26]. Un défaut similaire peut être observé dans des lignées lymphoblastoïdes établies à partir des lymphocytes de ces patients [24,26,27]. En revanche, des lignées lymphoblastoïdes dont le gène RPS24 est altéré montrent un taux élevé d'ARN 30S [27], alors que c'est la maturation des ARNr 5,8S et 28S qui est retardée en présence de la mutation de RPL35A, RPL5 et RPL11 [16,18].…”