UCLA

Ribosomes are large complex molecular machineries required for the synthesis of proteins via a process called protein translation. The importance of this molecular machine is conserved and is found within all living cells. Two subunits (designated the small and large subunits) form a functional ribosome. Each subunit is a complex of ribosomal RNA and a variety of proteins. Within the ribosome, amino acids are linked together depending on the order specified by the mRNA molecule.

Due to the importance of ribosomes in maintaining cellular function, one would expect defects in ribosome function, or biogenesis, to have a systemic effect on the organism. However, what is typically seen with disorders involving aberrant ribosome functions, a.k.a. ribosomopathy, is a tissue-specific defect. Specifically, patients normally present themselves with bone marrow failure, which ultimately leads to severe anemia. Other common features in patients include a shorter stature and a higher predisposition to certain cancers.

Diamond Blackfan anemia (DBA) is the first described ribosome-associated disorder with ribosomal protein S19, RPS19, being the most commonly mutated gene found in patients. Since then, the number of ribosomal proteins found mutated in DBA patients has expanded and includes genes such as RPL11, RPL5, and RPS24. Another ribosomopathy is 5q-syndrome. In this disease, a large chromosomal deletion is found in patients. Within the commonly deleted region (CDR), it is believe that ribosomal protein S14, RPS14, is the candidate gene associated with the macrocytic anemia seen in the disease.

The effects of p53 signaling during ribosome stress have been well documented in both patient bone marrow and in models of ribosomopathies. Aberrant ribosome function has been shown to increase p53 signaling through stabilization of p53 through a RP-MDM2-p53 checkpoint mechanism in the cell. Upon increases in p53 activity, p53-mediated cell cycle arrest and apoptosis occurs. Reversal of this increase in p53 signaling has been shown to relive the phenotypic and anemic defects in cellular and animal models. However, the use of p53 inhibition towards patient care has been cautioned due to risk of cancer progression. p53 is a major tumor suppressor gene in the cell and abolishing its activity has been shown to elevate cancer risk. Patients with malfunctioning ribosome proteins are at a higher risk of tumor development; therefore, targeting p53 may further enhance tumor risk. This dilemma raises the need for novel therapeutics in tackling the treatment of ribosomopathies and to further understand disease pathology.

Animal models are indispensible for understanding the molecular mechanism and pathology of a disease. Zebrafish is an attractive vertebrate model to use in biological studies due to its rapid development, small size, transparent nature, and ease of manipulating gene function. Furthermore, there are many conserved genes between mammals and zebrafish, thereby making the model useful for understanding gene function. The advent of genome-editing technologies applied to the zebrafish model (i.e. TALEN, CRISPR/Cas9) has expanded the use of the zebrafish to model human diseases.

In this work, we model two ribosome-associated disorders in the zebrafish, DBA and 5q-sydrome, via targeted RPL11 and RPS14, respectively. Using these models, we show that a delay in late-stage erythropoiesis occurs and is followed by cell death upon ribosome stress. The delay in late-stage erythropoiesis is independent of p53 signaling and may, in part, be due to overexpression of Lft1. We demonstrated the ability of RAP-011, an activin receptor type IIA ligand trap, to increase erythropoiesis in the zebrafish and restore erythroid levels in our zebrafish models. Furthermore, the effects of RAP-011 are independent of p53 signaling, and thus, offer a new strategy in the treatment of ribosomopathies.