Abstract
Ribosomal biogenesis is a fundamental process necessary for cell growth and division. Ribosomal protein L5 (Rpl5) is part of the large ribosomal subunit. Mutations in this protein have been associated with the congenital disease Diamond Blackfan anemia (DBA), a so called ribosomopathy. Despite of the ubiquitous need of ribosomes, clinical manifestations of DBA include tissue-specific symptoms, e.g., craniofacial malformations, eye abnormalities, skin pigmentation failure, cardiac defects or liver cirrhosis. Here, we made use of the vertebrate model organism Xenopus laevis and showed a specific expression of rpl5 in the developing anterior tissue correlating with tissues affected in ribosomopathies. Upon Rpl5 knockdown using an antisense-based morpholino oligonucleotide approach, we showed different phenotypes affecting anterior tissue, i.e., defective cranial cartilage, malformed eyes, and microcephaly. Hence, the observed phenotypes in Xenopus laevis resemble the clinical manifestations of DBA. Analyses of the underlying molecular basis revealed that the expression of several marker genes of neural crest, eye, and brain are decreased during induction and differentiation of the respective tissue. Furthermore, Rpl5 knockdown led to decreased cell proliferation and increased cell apoptosis during early embryogenesis. Investigating the molecular mechanisms underlying Rpl5 function revealed a more than additive effect between either loss of function of Rpl5 and loss of function of c-Myc or loss of function of Rpl5 and gain of function of Tp53, suggesting a common signaling pathway of these proteins. The co-injection of the apoptosis blocking molecule Bcl2 resulted in a partial rescue of the eye phenotype, supporting the hypothesis that apoptosis is one main reason for the phenotypes occurring upon Rpl5 knockdown. With this study, we are able to shed more light on the still poorly understood molecular background of ribosomopathies.
Highlights
whole mount in situ hybridization (WMISH) with an rpl5-specific antisense probe was performed at various developmental stages starting after gastrulation throughout late tailbud stages to investigate the spatiotemporal expression of rpl5 (Figures 1B–J)
The ribosomal protein ribosomal protein L5 (Rpl5) has been shown to be required for proper development of anterior organs and tissues such as the eyes, the brain, and the cranial cartilage in Xenopus laevis
The Rpl5 knockdown led to increased apoptosis and decreased proliferation
Summary
Cell growth and division are fundamental for the development of any multicellular organism These processes are highly regulated and during embryogenesis, each dividing cell requires an adequate number of ribosomes to cope with the demand for translation. This demand is ensured by ribosome biogenesis, which mainly takes place in the nucleolus and nucleus. Defects in ribosome biogenesis can lead to congenital diseases called ribosomopathies (Narla and Ebert, 2010; Farley-Barnes et al, 2019; Kang et al, 2021). Ribosomopathies such as the Diamond Blackfan anemia (DBA) or the Shwachman Diamond syndrome include various clinical manifestations. Several genes have been identified whose mutations lead to impaired pre-rRNA transcription, pre-rRNA processing, or ribosome assembly (Valdez et al, 2004; Weaver et al, 2015; Jenkinson et al, 2016; Kostjukovits et al, 2017; Warren, 2018)
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