The Ribosomal Protein L5 Functions During Xenopus Anterior Development Through Apoptotic Pathways

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.

Functions of block of proliferation 1 during anterior development in Xenopus laevis

“…Previous studies showed that upon knockdown of ribosomal proteins or factors, proliferative and apoptotic pathways are impaired [ 17 , 19 , 52 , 53 ]. Therefore, we investigated the number of proliferative cells via pH3 staining and apoptotic cells using TUNEL staining at stage 23.…”

“…In a previous study investigating the ribosomal protein L5 (Rpl5), we have shown that the expression of c-myc , which is an important player during ribosomal biogenesis as it enhances the performance of all three polymerases I-III, is reduced in embryos upon rpl5 knockdown. Furthermore, the co-injection of c-myc RNA partially rescued the eye phenotype which occurred upon rpl5 MO injection [ 53 ]. Additionally, Bellmeyer and colleagues showed that c-myc knockdown results in a deformed and smaller cranial cartilage similar to the here observed phenotype [ 54 ].…”

“…The knockdown of the ribosomal biogenesis factor nucleolar protein 11 leads to a late craniofacial cartilage malformation [ 52 ]. Recently, we also have shown that loss of Rpl5 leads to craniofacial cartilage as well as eye defects [ 53 ]. In summary, suppression of these investigated ribosomal biogenesis factors (as shown in earlier studies) results in comparable phenotypes as shown for Bop1 in this study, i.e., a disrupted development of the cartilaginous head structures, the brain, and the eye.…”

“…This is not in line with the fact that ribosomes are crucial for cell proliferation. In a previous study, we were able to partially rescue an eye phenotype, which occurred upon knockdown of a ribosomal protein, with c-myc RNA [ 53 ]. Hence, we analyzed whether c-myc RNA, as transcriptional enhancer important for proliferation, can rescue the bop1 MO-induced phenotype.…”

Bop1 is required to establish precursor domains of craniofacial tissues

“…It is well established that when mutated, factors that are involved in ribosome biogenesis can lead to significant craniofacial dysmorphologies in experimental models and human patients (Falcon et al, 2022;Griffin et al, 2015;Schreiner et al, 2022;Zhou et al, 2014). However, there is evidence that some of the proteins known to be necessary for ribosomal synthesis also have other cellular functions, particularly during early development (Bugner et al, 2011;Gessert et al, 2007;Tecza et al, 2011).…”