Project Summary/Abstract Ribosomopathies comprise a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function. Despite the need for ribosomes in all cell types, ribosomopathies display specific clin- ical phenotypes. Human Isolated Congenital Asplenia (ICA), which manifests at birth as absence of the spleen, or as hypoplastic spleen, without additional developmental anomalies, has been recently classified as a ri- bosomopathy. Given the central role of the spleen in host defense, ICA patients are vulnerable to life- threatening blood-borne infections. Germline heterozygous mutations were identified in the gene encoding RPSA [ribosomal protein (RP) SA; a component of the ribosome small subunit] in a large proportion of ICA pa- tients. The critical role for RPSA in human spleen development was surprising, given that mutations in other RPs had never been associated with asplenia. While spleen development constitutes an ideal system to study specific functions of RPs, animal models generated so far cannot be used to determine how deficiency of RPSA affects spleen development. Indeed, homozygosity for a null Rpsa allele causes early lethality in utero, while heterozygosity is not associated with asplenia. In order to overcome these limitations and to identify RPSA spleen-specific functions, we have generated mouse embryonic spleen cells with deficiency of either Rpsa or Rps14 (of note, haploinsufficiency of RPS14 causes impaired erythropoiesis in humans, without spleen defects). Preliminary results show that cell proliferation and protein synthesis are more strikingly re- duced in Rpsa versus Rps14 mutant splenic cell lines. Yet, little is known about the mechanisms underlying the specificity of RPSA-dependent phenotypes in mouse spleen cells and the potential conservation of these mechanisms in humans. We hypothesize that RPSA plays “specialized” roles that are restricted only to spleen development in mouse and humans. We plan to test this hypothesis via these specific aims: 1. To assess specificity of RPSA haploinsufficiency in a tractable murine spleen model system. By using unbiased genome-wide approaches (Ribo-seq), we will assess translation dynamics and we will measure actively trans- lated mRNAs in wildtype and mutant (deficient for Rpsa and Rps14, respectively) mouse embryonic spleen mesenchymal cells. These experiments will define RPSA-dependent mechanisms and translational targets that render spleen embryonic cells more susceptible to loss of RPSA versus RPS14. 2. To assess whether RPSA functions are conserved in a human spleen model system. We will generate and characterize human splenic cell cultures with RPSA deficiency by performing genome editing of RPSA in splenic mesenchymal cells obtained from human fetuses. These experiments will determine whether RPSA-dependent defects in mouse embryonic spleen cells, identified in Aim1, are conserved in human fetal spleen cells. This research will be g...