Gene expression regulation of human hemojuvelin by two small open reading frames
Year: 2012
Type: dissertation
Abstract: Iron is an essential element for many biological reactions carried out by living systems. A tight regulation of the systemic iron homeostasis is crucial to avoid pathological conditions of iron deficiency or overload. Juvenile hemochromatosis, is an early-onset inherited disorder associated to an iron overload caused by mutations on the hepcidin gene or in the gene encoding hemojuvelin (HJV). HJV is a glycosylphosphatidylinositol (GPI)-linked membrane protein shown to be a co-receptor for a class of ligands called bone morphogenetic proteins (BMPs), which trigger a response to the iron increase by activating the hepcidin transcription. Thus, HJV is involved on iron homeostasis through regulation of hepcidin transcription levels. A better knowledge of the mechanisms implicated in HJV gene expression is crucial to understand its role in the iron homeostasis. The 5’ leader sequence of the human HJV mRNA has two upstream AUGs (uAUGs) that share the same codon stop, forming two upstream open reading frames (uORFs) with 28 and 19 codons. To evaluate the effect of these uORFs in the translational regulation of HJV, reporter constructs containing several HJV 5’ leader sequences fused to the firefly Luciferase cistron, were tested in HeLa and HepG2 cells. Luciferase activity was measured by luminometry assays and normalized to the corresponding mRNA levels, quantified by real-time reverse transcription quantitative polymerase chain reaction (RT-PCR), to obtain translation efficiencies. The results revealed that the HJV uORFs decrease the translational efficiency of the main ORF in about 6-fold. Furthermore, we have observed that the HJV mRNA has a low leaky scanning ability that contributes to the translational repression of the main ORF. Thus, translation reinitiation is the main mechanism involved in the production of HJV protein. Aiming to further characterize the mechanism through which the HJV uORFs affect downstream translation, we have observed that the uORF2 encoded peptide seems to cause ribosomal stalling, which also prevents translation of the downstream main ORF. Together, these results produce a framework for understanding how human HJV gene expression is fine-tuned controlled during translation.
Author: Cláudia Onofre
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W575237881