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Analysis of the interactome of RPS19 mutants

ABSTRACT: Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. To clarify the function of RPS19, we compare the interactome of RPS19 wilde type and their two mutant proteins: RPS19-R62W and RPS19- R101H. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. We found striking differences between the interactomes of wild type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control.

INSTRUMENT(S): 6340 Ion Trap LC/MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Marianna Caterino

LAB HEAD: Margherita Ruoppolo

PROVIDER: PXD000640 | Pride | 2014-08-11

REPOSITORIES: Pride

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Analysis of the interactome of ribosomal protein S19 mutants.

Caterino Marianna M Aspesi Anna A Pavesi Elisa E Imperlini Esther E Pagnozzi Daniela D Ingenito Laura L Santoro Claudio C Dianzani Irma I Ruoppolo Margherita M

Proteomics 20140918 20

Diamond-Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one-fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild-type protein one, using the same functional proteomic approach that we employ ...[more]

PMID: 25069755

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Mechanism of action of RPS19R62W mutation in Diamond-Blackfan Anemia (DBA)

Project description:RPS19 mutations are the most common cause of the human disorder Diamond Blackfan Anemia. The R62W mutation was hypothesized to act in a dominant negative fashion and mice expressing RPS19R62W have many of the characteristics of Diamond Blackfan Anemia. Diamond-Blackfan Anemia (DBA), is an inherited erythroblastopenia associated with mutations in at least 8 different ribosomal protein genes. Mutations in the gene encoding Ribosomal Protein S19 (RPS19) have been identified in ~25% of DBA families. Most of these mutations disrupt either the translation or stability of the RPS19 protein and are predicted to cause DBA by haploinsufficiency. However, approximately ~30% of RPS19 mutations are missense mutations that do not alter the stability of the RPS19 protein and are hypothesized to act by a dominant negative mechanism. To formally test this hypothesis, we generated a transgenic mouse model expressing an RPS19 mutation in which an Arginine residue is replaced with a Tryptophan residue at codon 62 (RPS19R62W). Constitutive expression of RPS19R62W in developing mice was lethal. Conditional expression of RPS19R62W resulted in growth retardation, a mild anemia with reduced numbers of erythroid progenitors and significant inhibition of terminal erythroid maturation, similar to DBA. RNA profiling demonstrated over 700 dysregulated genes belonging to the same pathways that are disrupted in RNA profiles of DBA patient cells. The samples compared are RNA extracted from CD71 positive erythroblasts sorted from the bone marrow of wild type mice and mice expressing a mutant RPS19 (RPS19R62W) transgene

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Project description:Diamond-Blackfan anemia (DBA) is a congenital disorder that results predominantly from mutations in various ribosomal protein genes. In order to determine how these mutations affect the translation of specific mRNAs, we performed microarray analysis of polysomal transcripts isolated from lymphoblast cells derived from DBA patients carrying different haploinsufficient mutations in either RPS19 or RPL11. One of the few overlapping transcripts that we found significantly decreased on the polysomes codes for branched-chain aminotransferase-1 (BCAT-1). We go on to determine that translation of BCAT-1 is especially impaired in cells carrying mutations in the small ribosomal protein genes, and provide evidence that this effect is due to its unusually long 5’UTR. The BCAT-1 enzyme is critical for synthesis of the branched-chain amino acids, including leucine. It is known that DBA patients respond well to orally administered leucine, and large-scale clinical trials as such are currently underway. Our results suggest that the haploinsufficient loss of ribosomal protein genes results in a decrease of BCAT-1 translation and provide an explanation for why leucine administration is beneficial for DBA patients.

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Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

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