Genetic manipulation of RPS5 gene expression modulates the initiation of commitment of MEL cells to erythroid maturation: Implications in understanding ribosomopathies

Abstract

Impairment of ribosome biogenesis contributes to the molecular pathophysiology of ribosomopathies by deregulating cell-lineage specific proliferation, differentiation and apoptosis decisions of haematopoietic progenitor cells. Here, using pro-erythroblast-like murine erythroleukemia (MEL) cells, a model system of erythroid maturation, we aimed to investigate whether genetic manipulation of RPS5 expression affects the capacity of cells to grow and differentiate in culture. Parental MEL cells stably transfected with full length RPS5 cDNA in sense (MEL-C14 culture) or antisense (MEL-antisenseRPS5 culture) orientation, as well as MEL cells transiently transfected with siRNAs specific for RPS5 gene silencing (MEL-RPS5siRNA culture) were assessed for their ability to fully execute their erythroid maturation program in culture. The data obtained thus far indicate that: a) MEL-antisenseRPS5 exhibit a pronounced delay in the initiation of differentiation, as well as an impairment of commitment, since the continuous presence of the inducer in culture is required for the cells to fully execute their erythroid maturation program. b) RNAi-mediating silencing of RPS5 gene expression resulted in the inability of MEL cells to differentiate; however, when these cells were allowed to recapitulate normal RPS5 gene expression levels they regained their differentiation capacity by accumulating high proportion of erythroid mature cells. c) Interestingly the latter, is accompanied by morphological changes of cells and an impairment of their proliferation and apoptosis potential. Such data for the first time correlate the RPS5 gene expression levels with the differentiation capacity of MEL cells in vitro, a fact that might also have implications in understanding ribosomopathies.

miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis

“…Studies over the past years have shown that there is stringent and precise regulation of ribosome levels and function during erythropoiesis. This is evident under conditions of mild ribosome dysfunction, as it occurs in a group of disorders termed ribosomopathies [14,44,45]. The latter are caused by heterozygous mutations in ribosomal proteins and associate with highly specific symptoms.…”

“…As with other cancer types, erythroleukemias show an imbalance in favor of hyperproliferative phenotypes over differentiation. However, extensive work in established cell line erythroleukemia models (especially murine erythroleukemia, MEL, cells) has shown that the leukemic phenotype can be reversed in vitro by reintroducing the cells into the path of differentiation [10][11][12][13][14][15][16]. Differentiation is initiated after treatment with chemical inducers (such as hexamethylene bisacetamide, HMBA) and involves an extensive yet precisely orchestrated gene expression program [10,11,17].…”

Roles of EvpP in Edwardsiella piscicida-Macrophage Interactions

“…The increasingly accumulated evidence suggests that ribosomal proteins has extra-ribosomal functions associated with cell differentiation, apoptosis, regulation of signal pathway, and DNA repair (LindstrÖm, 2009;Warner and Mcintosh, 2009). RPS5 was also found to participate in extra-ribosomal activities such as cell differentiation (Matragkou et al, 2008;Vizirianakis et al, 2015), intracellular trafficking (Matragkou et al, 2009), and hepatic fibrosis (Xu et al, 2016).…”

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

“…Mouse erythroleukemic (MEL) cells (clone 745-PC–4) were derived from the MEL 745 cell line, originally isolated by Friend et al [ 25 ] and were maintained and induced to differentiate with 5mM of HMBA (N,N-Hexamethylene-bis- acetamide, Sigma) as previously described [ 26 ]. Cell culture density and differentiation (hemoglobin production) were assessed as previously described [ 26 ].…”