Ribosomal protein dysfunction causes abnormal ribosomal particle assembly and affects protein synthesis in all cell types. Although RP dysfunction affects multiple tissues and systems, pathological defects in hematopoietic systems, such as aberrant development in Diamond–Blackfan anemia (DBA), are the most frequently observed phenotypes [1
Mutations in RP genes reduce the efficiency of ribosome biogenesis, which is indispensable for immature erythrocytes in early and rapid growth phases [4
]. Defective ribosome biosynthesis leads to excessive release of RPs such as RPL5 and RPL11, which may increase p53 activity by activating the RPS MDM2-p53 signaling pathway [5
]. Because erythroid progenitor cells are highly sensitive to p53 activation [6
], subsequent activation of the p53 pathway in erythroid progenitor cells leads to cell cycle arrest and apoptosis and finally to hematopoietic disorders. Interestingly, these hematopoietic defects were rescued by increased RP expression or reduced p53 activity in zebrafish and in erythroid progenitor cells from DBA patients [7
]. It was also observed that Rpl22l1 plays essential roles in hematopoiesis, but acts independently of p53 and does not induce apoptosis [11
DBA is a rare congenital red blood cell dysplasia that develops soon after birth. This disorder is mainly characterized by reduced reticulocyte counts, selective red cell aplasia, and macrocytosis, while a number of other blood cell lineages, such as neutrophils and platelets, are maintained or slightly decreased. Furthermore, DBA patients suffer other developmental defects, such as short stature, cleft lip, hand deformities, kidney and heart hypoplasia, and tumor predisposition [12
]. Ribosome biogenesis defects have been identified as a major cause of DBA, and mutations of genes encoding RPs have emerged as the main cause of DBA. Indeed, various mutations have been found in coding and noncoding regions of at least 13 RPs, including RPL5, RPS7, RPL9, RPS10, RPL11, RPS15, RPS17, RPS19, RPS24, RPS26, RPL36, RPS27a
, and RPL35a
, accounting for about 55% of all DBA cases [15
was the first identified causative gene in DBA patients, and its mutations account for 25% of DBA cases [24
RPL11 is one of 79 vertebrate RPs, and its gene mutations occur on coding and intronic binding regions of chromosome 1 [17
]. DBA patients with mutations in RPL11
show typical hematopoietic defects, and 67% of these have physical deformities, especially of the thumb [17
]. In particular, RPL11 dysfunction results in abnormal erythrocyte development, markedly decreased progenitor cell proliferation, delayed erythroid differentiation, and markedly increased apoptosis [25
]. Importantly, these are different from the deficiencies in DBA patients with RPS19
]. Hence, divergent mechanisms underlie hematopoietic defects in DBA patients with different RP mutations. Moreover, RPL11 dysfunction in erythroblasts lead to aberrant erythroblast differentiation, reduced hemoglobin, and unusual cell morphology [26
Zebrafish are a classic animal model for mechanistic studies of embryonic development, hematopoiesis, and DBA [27
]. Knockdown of most RP genes causes developmental defects in the brain of zebrafish, indicating that the vast majority of RP functions are associated with nervous system development [29
]. Several reports focus on regulation of zebrafish hematopoiesis by the RP proteins Rps7, Rpl11, Rps19, and Rps29 [8
]. Rpl11 dysfunction in zebrafish embryos leads to defective development, hematopoiesis, brain development, and shape dysplasia [8
]. Furthermore, Danilova et al.
reported that the hematopoietic defects caused by Rpl11 dysfunction were fully reversed by the inhibition of p53 activity in zebrafish mutants [8
], indicating that p53 may regulate Rpl11 in zebrafish [25
]. However, hematopoiesis in zebrafish with dysfunctional Rps19 is not fully recovered by inhibition of p53 [32
]. Presumably, these varying phenotypic defects in zebrafish embryos with dysfunctional Rpl11 are regulated by multifactorial interactions of genes, regulatory networks, and signaling pathways.
High-throughput transcriptome sequencing is now widely accepted as a useful tool for investigating human disease mechanisms and gene functions. However, this technology has not been widely used in zebrafish research. Using microarray technology, Danilova et al. (2011) studied the impact of Rpl11 deficiency on hematopoiesis of zebrafish embryos. However, because of the limitations of detectable gene transcripts, some important aspects of Rpl11 functions in zebrafish may only be elucidated using high-throughput sequencing.
In this study, we examined genome-wide transcription by high-throughput transcriptome sequencing of Rpl11-deficient zebrafish embryos. In combination with bioinformatic analyses, we studied affected genes, hematopoiesis-related signaling pathways, and molecular regulatory networks in Rpl11-deficient zebrafish embryos. We found that Rpl11 deficiency leads to defects of hemoglobin synthesis and hematopoiesis, which were caused by dysregulation of iron metabolism-related genes and impaired HSC differentiation and proliferation. These analyses provide new insights into the pathogenesis of mutant Rpl11-mediated DBA.