Browsing by Author "Martins, Rute"
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- Alternative polyadenylation and nonsense-mediated decay coordinately regulate the human HFE mRNA levelsPublication . Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, LuísaNonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 39 untranslated region (UTR), along with exon seven. Therefore, this 39 UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 39-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.
- Differential HFE Gene Expression is Regulated by Alternative Splicing in Human TissuesPublication . Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, PaulaBackground - The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings - Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance - HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.
- Duodenal Cytochrome B and Hephaestin Expression is Regulated by the Soluble HFE IsoformPublication . Silva, Bruno; Martins, Rute; Proença, Daniela; Faustino, PaulaINTRODUCTION: Hereditary Hemochromatosis is an autosomal recessive disorder characterized by excessive intestinal iron absorption and iron deposition in organs such as liver, heart and pancreas, potentially leading to cirrhosis, hepatocelular carcinoma, diabetes, cardiac failure and arthritis. This disorder is mainly due to mutations in HFE gene. HFE protein associates with beta-2 microglobulin (B2M) for trafficking to the cell surface. However, the HFE’s role on iron homeostasis is not completely cleared. It may regulate hepcidin expression in the liver and iron trafficking in the duodenum. Several HFE alternative splicing transcripts have been reported, but their structural and functional characterization have been poorly studied. MATERIALS AND METHODS: Aiming to investigate the putative biological role of an alternative HFE transcript originated by the intron 4 inclusion, we measured its expression level in several human tissues by quantitative Real-Time PCR. Also, we produced the corresponding GFP-tagged HFE variant. HepG2 cells were transfected with this construct and protein cellular location analyzed by immunofluorescence, using B2M, TfR1 and calnexin antibodies. In parallel, immunoprecipitation was performed. Finally the intron 4 inclusion variant was over-expressed in a human duodenum adenocarcinoma cell line (Hutu-80) under normal and iron overload conditions and the expression of several iron metabolism genes (TFR1, DMT1, DCYTB, SLC40A1 and HEPH) evaluated by quantitative Real-Time PCR. RESULTS: We have found that the intron 4 inclusion transcript has an ubiquitous expression in the analyzed tissues, being its relative expression higher in duodenum and lower in the liver. Also, we found that this variant gives rise to a truncated protein (sHFE) that is secreted by the cells and is able to maintain its interaction with B2M. Its overexpression in HuTu-80 cells showed that sHFE down-regulates the duodenal cytochrome b (CYBRD1) expression in about 20% independently of cellular iron status, as it happens with the HFE_full length protein. Also, sHFE seems to be involved in the down-regulation of hephaestin (HEPH) expression, being its effect higher in the presence of iron overload (reduction of ~40 and ~50%, respectively). CONCLUSIONS: Through this study we might have unveiled the contribution of the HFE’s intron 4 inclusion splice variant to the maintenance of iron homeostasis. sHFE may be secreted into the bloodstream and act in remote tissues such as the duodenum, down-regulating the expression of some of the iron metabolism related genes, as CYBRD1 and HEPH, and consequently reducing dietary iron absorption. Also we are currently exploring the hypothesis of a possible effect of sHFE in the expression of other iron metabolism related genes in hepatic cells and macrophages.
- Early modification of sickle cell disease clinical course by UDP-glucuronosyltransferase 1A1 gene promoter polymorphismPublication . Martins, Rute; Morais, Anabela; Dias, Alexandra; Soares, Isabel; Rolão, Cristiana; Ducla-Soares, JL; Braga, Lígia; Seixas, Teresa; Nunes, Baltazar; Olim, Gabriel; Romão, Luísa; Lavinha, João; Faustino, PaulaElevated erythrocyte destruction in sickle cell disease (SCD) results in chronic hyperbilirubinaemia and, in a subset of patients, cholelithiasis occurs. We investigated whether the (TA)n promoter polymorphism in the UDP-glucuronosyltransferase 1A1 gene (UGT1A1) may modify bilirubin metabolism, influencing bilirubinaemia, predisposition to cholelithiasis and subsequent cholecystectomy, in a group of 153 young SCD patients (mean age 12.0 +/- 9.0 years) predominantly of Bantu beta S haplotype. The concomitant effect of alpha thalassaemia was also analysed. Among the several UGT1A1 genotypes found, the most frequent were the (TA)6/(TA)6 (n = 37), (TA)6/(TA)7 (n = 60) and (TA)7/(TA)7 (n = 29). These groups of patients did not significantly differ in age, gender ratio and haemoglobin, foetal haemoglobin and reticulocyte levels. On the other hand, total bilirubin levels were significantly different between groups, with an increased (TA) repeat number being associated with higher bilirubinaemia. Furthermore, both cholelithiasis and cholecystectomy were more frequent in groups with higher (TA) repeat number, although the former association was not statistically significant. None of the mentioned parameters is statistically different within UGT1A1 groups with the presence of alpha thalassaemia. Thus, the UGT1A1 promoter polymorphism may represent an important nonglobin genetic modifier of Bantu SCD patients' clinical manifestations, even at a young age.
- Interaction between HFE and haptoglobin polymorphisms and its relation with plasma glutathione levels in obese childrenPublication . Laura, Aguiar; Marinho, Cláudia; Martins, Rute; Alho, Irina; Ferreira, Joana; Quinhones Levy, Pilar; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaObesity among children has emerged as a serious public health problem. The growing prevalence of childhood obesity has led to the appearance of serious complications, including a chronic systemic inflammation associated with oxidative stress. In the present study, we analysed the interaction between two genes related with iron metabolism - HFE and haptoglobin – and the plasmatic concentration of glutathione, as a way to evaluate the antioxidant response capacity in obesity. To achieve this, 118 obese children and 89 eutrophic children were recruited for the study. Results showed that although obese children present a significantly decreased tGSH levels, once we analysed separately children based on their haptoglobin phenotype, the decreased tGSH levels is significant only for the Hp 2 allele. Additionally, Hp 2.2 obese children carrying H63D polymorphism show significantly lower tGSH/GSSG values. Our results found an association of haptoglobin and HFE with oxidative stress in childhood obesity.
- Post-transcriptional regulation of HFE gene expressionPublication . Martins, Rute; Faustino, Paula; Romão, LuísaIron is a key element for numerous metabolic processes in living cells. The challenge for most organisms is to acquire the adequate amounts of iron for these processes yet avoiding the toxicity associated with free iron. In fact, disruptions of iron homeostasis account for some of the most common human diseases. Amongst these, lays hereditary hemochromatosis, which is mainly associated with mutations in the HFE gene, leading to iron overload in specific organs. HFE protein acts in iron homeostasis by regulating the expression of hepcidin in the liver. Besides the major HFE transcript, which is expressed at low levels in a wide range of human tissues, several additional alternative HFE transcripts have been described. The work presented in this dissertation addresses the characterization of HFE alternative transcripts, the biological mechanisms involved in their genesis as well as their physiological significance and regulation. A variety of human tissues was analysed and shown to express several alternatively spliced HFE transcripts. Functional analysis of the corresponding proteins revealed that alternative splicing can either generate non-functional HFE protein variants or produce a soluble HFE variant that is secreted by cells associated with beta2-microglobulin. This soluble HFE may have a vital role in iron homeostasis by acting as an agonist or antagonist of the full length HFE. Furthermore, HFE transcripts were found to be physiologically regulated by the nonsense-mediated mRNA decay (NMD), since its levels are significantly increased when depleting human cells from a key NMD effector. Through the analysis of several human tissues, it is shown that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites. This tissue-specific polyadenylation pattern seems to respond to cellular iron status, acting coordinately with NMD to fine-tune HFE’s expression levels. The regulation of HFE gene expression is here shown to be post-transcriptionally influenced by alternative splicing, alternative polyadenylation and nonsense-mediated mRNA decay mechanisms. These findings may hint future directions in the active field of iron biology research and provide interesting cues that could be translated into new therapeutics for iron homeostasis disorders through the HFE-mediated regulation of hepcidin.
- A soluble HFE splice variant seems to regulate the expression of duodenal cytochrome b and hephaestin contributing to iron metabolism regulationPublication . Silva, Bruno; Martins, Rute; Proença, Daniela; Faustino, PaulaINTRODUCTION: Hereditary Hemochromatosis is an autosomal recessive disorder characterized by excessive intestinal iron absorption and pathological iron deposition in organs such as liver, heart and pancreas. The disease is predominantly caused by homozygosity for the p.C282Y mutation in HFE, which impairs protein association with its chaperone beta-2 microglobulin (B2M), for correct folding and traffic to the cell surface. Several alternative HFE transcripts have been reported but their functional significance remains elusive. Since we have identified an alternative HFE transcript due to intron 4 inclusion, we aimed to investigate its physiological role on iron homeostasis. MATERIALS AND METHODS: We analysed the expression level of the alternative transcript in several human tissues by quantitative Real-Time PCR. In addition, we produced the corresponding GFP-tagged HFE protein variant. HepG2 cells were transfected with this construct and protein cellular location analyzed by immunofluorescence using several antibodies. In parallel, immunoprecipitation was performed. Finally, the variant was over-expressed in a duodenal cell line (Hutu-80) under normal and iron overload conditions and the expression of several iron metabolism related genes (TFR1, DMT1, CYBRD1, SLC40A1 and HEPH) was evaluated by quantitative Real-Time PCR. RESULTS: We have found that the HFE_intron 4 inclusion transcript has an ubiquitous expression, being its relative expression higher in duodenum and lower in the liver. Also, we found that it gives rise to a truncated soluble protein (sHFE) that is secreted by cells maintaining its interaction with B2M. Its overexpression in HuTu-80 cells revealed that the sHFE is able to down-regulate the duodenal cytochrome b (CYBRD1) expression, as it happens with the HFE_full length protein. Also, it seems to play a specific role in the regulation of hephaestin (HEPH) expression. CONCLUSIONS: Through this study we might have unveiled the contribution of the sHFE variant to iron homeostasis. In fact, sHFE may be secreted into the bloodstream and act in remote tissues such as duodenum, down-regulating the expression of some of the iron metabolism related genes, as CYBRD1 and HEPH, and consequently reducing dietary iron absorption, preventing iron overload and contributing to iron metabolism regulation. Partially funded by FCT: PTDC/SAU-GMG/64494/2006; CIGMH; SFRH/BD/21340/2005 and SFRH/BD/60718/2009. The authors declare no competing interests.
- The functional significance of E277K and V295A HFE mutationsPublication . Silva, Bruno; Martins, Rute; Proença, Daniela; Fleming, Rita; Faustino, PaulaHereditary haemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption resulting in increased pathological body iron stores. It is typically associated with homozygosity for the c.845G>A (p.C282Y) mutation in the HFE gene. However, other HFE alterations have been reported in affected individuals but their association with the disease is unclear. This study analysed the functional consequences of two HFE mutations, c.829G>A (p.E277K) and c.884T>C(p.V295A). Firstly, it was shown that c.829G>A affects the HFE splicing by diminishing the full length HFE and ivs4_66bp inclusion transcript levels, while increasing the amount of exon 4 skipping transcript. Immunofluorescent techniques showed that the HFE_E277K protein had a diffuse distribution(similar to HFE_C282Y) while HFE_V295A presented at the cell surface and perinuclear compartments (resembling HFE_wt). Immunoprecipitation assays revealed a decreased association of HFE_E277K and HFE_V295A with both b2-microglobulin (B2M; 38 ± 7% and 66 ± 8%, respectively) and transferrin receptor (TFRC, also termed TFR1) (58 ± 2% and 49 ± 16%, respectively). Herein, we prove that both mutations partially abrogate HFE association with B2M and TFRC, crucial for its correct processing and cell surface presentation. Although E277K has a more deleterious effect than V295A, we propose that both mutations may play a role in the development of hereditary haemochromatosis.