Hypermethylation of 28S ribosomal RNA in β-thalassemia trait carriers

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Abstract

Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2′-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of β-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in β-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in β-thalassemia trait carriers.

Introduction

β-thalassemia refers to the hereditary blood disorder characterized by reduced (β+) or absent (β0) beta globin chains and more than 200 different point mutations in addition to cases of gross gene deletions have been characterized to date as being associated with β-thalassemia [22]. The reduced or absent expression of the β-globin chain leads to a loss of balanced globin chain synthesis and the excess α-globin chains precipitate in the cell resulting in ineffective erythropoiesis and increased hemolysis [15]. Collectively the loss of functional hemoglobin presents as an anemia of variable severity that is dependent upon a number of factors including the co-inheritance of α-globinopathies [20]. Heterozygous β-thalassemia generally presents as an asymptomatic to intermediate phenotype, while compound heterozygote β0-thalassemia typically present as a severe, transfusion dependent condition [22].

Mature red blood cells arise from hematopoetic stem cells predominantly resident in the bone marrow in a process that is controlled by the action of a number of cytokines including erythropoietin (EPO). In β-thalassemia the developmental process is believed to be inappropriately terminated by the induction of apoptosis as a consequence of precipitation of the unpaired α-globin chains in the process termed ineffective erythropoiesis [15]. The resulting anemia promotes the increased production of EPO, leading to expansion of the bone marrow mass [3]. Critically however, some evidence has suggested that erythroid progenitor cells in β-thalassemia patients may have an inherently increased expansion capacity, modulated at least in part by the hyperphosphorylation of ERK1/2 [21], a protein that regulates the expression of a large number of genes both directly and indirectly. Studies have shown that ERK1/2 plays a role in enhancing Myc activity by increasing stabilization of Myc through phosphorylation of Ser 62 resulting in a decrease of Myc ubiquitin mediated degradation [16]. The Myc family of transcription factors regulate the expression of thousands of genes that control a wide range of cellular processes including transcription, translation, regulation of chromatin structure, DNA replication, and more recently, ribosome biogenesis [18].

Ribosome biogenesis involves more than 170 accessory factors and involves a number of transcriptional and post-transcriptional events [4]. Mature ribosomes consist of four different ribosomal RNA (rRNA) molecules together with some 80 different ribosomal proteins, and the rRNA molecules undergo extensive co- and post-transciptional modification including endo and exo-nuclease cleavage, as well as specific 2′-O-methylation and pseudouridylation [1]. 2′-O-methylation is regulated by box C/D snoRNAs (small nucleolar RNAs) in association with fibrillarin, while box H/ACA snoRNAs in association with dyskerin regulate pseudouridylation [7]. These biochemical modifications could finely tune the catalytic ribozyme activity of the rRNAs which directly control protein synthesis [1]. Indeed, evidence is emerging that these post-transcriptional modifications serve to modulate ribosomal assembly and translational capacity [24].

Defects in ribosome biogenesis have been associated with several inherited bone marrow failure syndromes including Diamond-Blackfan anemia [12] which has been shown to result from mutations in a number of ribosomal protein genes [19]. Given that β-thalassemia is the most common anemia worldwide, we sought to determine if there was evidence of altered ribosome biogenesis in β-thalassemia and additionally looked at hemoglobin E trait.

Section snippets

Sample collection and preparation

In this study, all blood samples were taken after obtaining individual informed consent under a protocol approved by the Committee for Human Rights related to Experimentation, Mahidol University. For experiments utilizing whole blood, peripheral blood was taken from healthy normal (2.5 ml) and heterozygous β-thalassemia donors (0.5–1.5 ml). All individuals donating specimens used as whole blood were screened as heterozygous β-thalassemia as previously described [8]. Normal controls were chosen on

Results and discussion

The post transcriptional modification of rRNA molecules by specific 2′-O-methylation and pseudouridylation are critical steps in ribosome biogenesis [7]. Until recently, determination of the methylation status of rRNA molecules was a time consuming and technically difficult process [14]. The application of RT-qPCR with high and low concentration dNTPs to determine methylation status is a relatively new methodology that considerably simplifies determination of comparative methylation status of

Authorship and disclosures

DRS and HCM designed the study. PJ, WS, SF and SS performed sample collection, genotyping and hemoglobin typing. PL and WS were responsible for all experimental work. DRS, HCM, JJD, SH, JE, PL, AF, and PJ were responsible for analysis and interpretation. DRS, WS HCM, JJD and PL wrote the manuscript with the approval from all authors. All authors declared no conflict of interest in this work.

Acknowledgements

The authors thank Nathalie Pion for technical help. This work was supported by a Research Chair Grant from the National Science and Technology Development Agency (NSTDA), the Thailand Research Fund (BRG5780004 and IRG5780009) and by INCa projet libre RIBOCAN from La Ligue National Contre le Cancer and Association pour la Recherche contre le Cancer. P.L. was supported by a Thai Royal Golden Jubilee Research Scholarship and the French Embassy in Thailand for part of the study. W.S. is supported

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  • Cited by (0)

    1

    WS and PL contributed equally to this work.

    2

    HCM and DRS contributed equally to this work.

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