The interplay between mRNA translation and nonsense-mediated decay in AUG-proximal nonsense-mutated transcripts

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The unified model for NMD, proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing residual downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD through a mechanism independent of translation re-initiation. In this seminar, I will focus on the mechanistic basis for this NMD resistance and how it involves the step of translation initiation

mRNA Metabolism in Health and Disease

Editorial.This article belongs to the Special Issue mRNA Metabolism in Health and Disease.Eukaryotic gene expression involves several interlinked steps, in which messenger RNAs (mRNAs), which code for proteins, are the key intermediates [...].info:eu-repo/semantics/publishedVersio

Determinants of household debt in Portugal

Household debt in Portugal has increased gradually, especially after the financial crisis of 2007-2008. In 2018, about 11 years after the crisis, there are still high values of household indebtedness. In this sence, this dissertation conducts an empirical investigation to assess the determinants of household debt in Portugal by performing a time series econometric analysis from the 1989 period up to 2018. An equation to test seven hypotheses (house prices, financial asset, falling wages, age structure, welfare retrenchment, low-interest rate, and expenditure cascade) was created and estimated in order to take into account all the hypothesis referred to in the literature that explain household indebtedness. Results show that wages are the most robust determinant both in the long-term and in the short-term. In the long-term, a decrease in wages causes an increase in Portuguese household debt. On the other hand, in the short-term an increase in wages causes an increase in Portuguese household debt. Also, for short-term, the house prices have a strong impact on the Portuguese household debt. Also with regard to long-term, the real long-term interest rate and welfare retrenchment have a significant impact on household debt.A dívida das famílias em Portugal aumentou gradualmente, especialmente após a crise financeira de 2007-2008. Em 2018, cerca de 11 anos após a crise, ainda existem altos valores de endividamento das famílias. Nesse sentido, esta dissertação realiza uma investigação empírica para avaliar os determinantes da dívida das famílias em Portugal, através de uma análise econométrica de séries temporais para o período entre 1989 e 2018. De forma a levar em consideração todas as hipóteses referidas na literatura que explicam o endividamento das famílias, foi construída e estimada uma equação para testar sete hipóteses (preços das casas, ativos financeiros, salários em queda, estrutura etária, redução do bem-estar, taxa de juro baixa e expenditure cascade). Os resultados obtidos indicam que o salário é o determinante mais robusto a longo e a curto prazo. A longo prazo, uma diminuição dos salários causa um aumento da dívida das famílias portuguesas. A curto prazo, um aumento nos salários causa um aumento na dívida das famílias portuguesas. Também a curto prazo, verifica-se que os preços das casas têm um forte impacto na dívida das famílias portuguesas. Relativamente a longo prazo, também se verifica que a taxa de juro e a redução do bem-estar social têm um impacto significativo na dívida das famílias

An upstream open reading frame regulates de translational efficiency of the human erythropoietin transcript

Among the various cis-acting elements present in the 5’ leader sequence of mRNAs there are upstream open reading frames (uORFs). Although their function is still poorly understood, they are known to downregulate the main ORF expression of several human transcripts that code for key regulatory genes. The human erythropoietin (EPO) is a glycoprotein that was initially characterized has a hormone mainly synthesized and released from the kidney, with a key role in hematopoiesis. However, many recent reports have implicated EPO in several non-hematopoietic functions and have shown its production in several other organs. Consequently, it might be used as a therapeutic target for the treatment of several human disorders. We found a natural occurring 14-codon-uORF on the human EPO transcript. Our belief is that understanding the molecular mechanisms through which the EPO uORF controls translation may be valuable in the determination of these EPO-based therapies. To explore the mechanisms by which EPO uORF controls translational efficiency, HepG2, HEK293 and REPC cells were transfected with several constructs carrying the luciferase reporter gene with the intact or disrupted EPO uORF, with or without the EPO 3’ untranslated region (3’UTR). Luciferase activity was measured by luminometry and normalized to the corresponding mRNA levels to obtain translation efficiencies. The mRNA levels were quantified by real-time RT-PCR. Furthermore, we also analyzed its response to several cell stress stimuli. Results show that the EPO uORF can decrease the main ORF translation efficiency in about 3-fold. In addition, our data support the conclusion that reinitiation, and in less extent leaky scanning, are responsible for the main ORF translation. In addition, the 3’UTR does not affect the role of the uORF, but it increases the luciferase levels, probably by stabilizing the mRNA. Specifically in REPC cells, translational inhibition mediated by the EPO uORF is overridden in response to chemical hypoxia, which is due to less recognition of the uAUG

Gene expression regulation by upstream open reading frames and human disease

Upstream open reading frames (uORFs) are major gene expression regulatory elements. In many eukaryotic mRNAs, one or more uORFs precede the initiation codon of the main coding region. Indeed, several studies have revealed that almost half of human transcripts present uORFs. Very interesting examples have shown that these uORFs can impact gene expression of the downstream main ORF by triggering mRNA decay or by regulating translation. Also, evidence from recent genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of many human diseases, including malignancies, metabolic or neurologic disorders, and inherited syndromes. In this review, we will briefly present the mechanisms through which uORFs regulate gene expression and how they can impact on the organism's response to different cell stress conditions. Then, we will emphasize the importance of these structures by illustrating, with specific examples, how disturbed uORF-mediated translational control can be involved in the etiology of human diseases, giving special importance to genotype-phenotype correlations. Identifying and studying more cases of uORF-altering mutations will help us to understand and establish genotype-phenotype associations, leading to advancements in diagnosis, prognosis, and treatment of many human disorders

The role of mRNA translation on nonsense-mediated decay inhibition in disorders due to nonsense mutations

Resumo de apresentação ora

Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

This article belongs to the Special Issue mRNA Metabolism in Health and Disease.ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5'-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5'-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.This research was partially funded by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). Joana Silva was acknowledged with financial support from a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/106081/2015).info:eu-repo/semantics/publishedVersio

Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the interaction of PABPC1 with the translation initiation complex

Apresentação oral por convite.Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing a premature termination codon (PTC). The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human b-globin mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. We have reported that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. In fact, we have shown that the establishment of an efficient translation termination reaction at the AUG-proximal PTC is dependent on PABPC1 interaction with the initiation factor eIF4G and with the release factor eRF3 at the terminating ribosome. These interactions underlie critical 3’-5’ linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation. Furthermore, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. This work corroborates a role for PABPC1 on NMD evasion of transcripts carrying an AUG-proximal PTC and provides further insights into the mechanistic details of PTC definition and translation initiation

Gene expression regulation by upstream open reading frames in rare diseases

Upstream open reading frames (uORFs) constitute a class of cis-acting elements that regulate translation initiation. Mutations or polymorphisms that alter, create or disrupt a uORF have been widely associated with several human disorders, including rare diseases. In this mini-review, we intend to highlight the mechanisms associated with the uORF-mediated translational regulation and describe recent examples of their deregulation in the etiology of human rare diseases. Additionally, we discuss new insights arising from ribosome profiling studies and reporter assays regarding uORF features and their intrinsic role in translational regulation. This type of knowledge is of most importance to design and implement new or improved diagnostic and/or treatment strategies for uORF-related human disorders.This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). JS and RF are supported by fellowships from Fundação para a Ciência e a Tecnologia (SFRH/BD/106081/2015 and SFRH/BD/114392/2016, respectively).info:eu-repo/semantics/publishedVersio

Translational control of the human hemojuvelin via upstream open reading frames

Iron is an essential element for many biological reactions carried out by living systems. A tight regulation of systemic iron homeostasis is crucial to avoid the pathological conditions of iron deficiency or overload. Juvenile hemochromatosis, is an early-onset inherited disorder associated to 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 class of ligands called bone morphogenetic proteins (BMPs). 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 (uORF) 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 and normalized to the corresponding mRNA levels, quantified by real-time 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, reinitiation is the mechanism mainly involved in the production of HJV protein. Aiming to characterize the mechanism through which the HJV uORFs affect downstream translation, we have observed that the amino acid sequences of the uORFs encoded peptides seem to cause ribosomal stalling, which also impede translation of the downstream main ORF