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

Publication . Onofre, Cláudia; Menezes, Juliane; Duarte, Andreia; Peixeiro, Isabel; Barbosa, Cristina; Costa, Paulo; Romão, Luísa

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.

2016-10Conference object

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How mRNA translation is involved in modulating nonsense-mediated decay in transcripts with short open reading frames

Publication . Onofre, Claudia; Menezes, Juliane; Peixeiro, Isabel; Costa, Nuno; Barbosa, Cristina; Romão, Luísa

Beyond its well-known hematopoietic action, erythropoietin (EPO) has diverse cellular effects in non-hematopoietic tissues. For example, in cases of tissue injury, such as cardiac ischemia or acute myocardial infarct, EPO expression increases locally, providing a cardioprotective effect. Cellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at translation initiation and is mediated via different cis-acting elements, including upstream open reading frames (uORFs). The human EPO 5’ untranslated region (5’UTR) has one uORF with 14 codons that is conserved among different species, indicating its potential regulatory role. To test whether EPO expression is translationally regulated in response to ischemia in cardiac tissue, reporter constructs containing the normal or mutant EPO 5’UTR fused to the Firefly luciferase cistron were expressed in H9c2 (heart/myocardium myoblasts) and C2C12 (muscle myoblasts) cell lines. Luminometry assays revealed that the EPO uORF represses translation of the main ORF at about 60-70%, in both cell lines. Under chemical ischemia, EPO uORF-mediated translation repression is specifically released in muscle cells. In response to hypoxia, translational derepression occurs in both cell lines. Although the eIF2-alpha phosphorylation occurs in both conditions, thapsigargin treatment does not affect EPO translation. We are currently exploring additional mechanisms through which EPO cardioprotection effects are regulated at the translational level.

2015-10-14Conference object

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

Publication . Onofre, Cláudia; Menezes, Juliane; Peixeiro, Isabel; Romão, Luísa

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTCs). Although NMD has been intensively studied, it is still poorly understood how NMD discriminates between PTCs and normal stop codons. The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmic 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. Here, we will present the mechanistic basis for this NMD resistance and how it involves the step of mRNA translation initiation.

2016-07Conference object

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Clinical utility of the polygenic LDL-C SNP score in familial hypercholesterolemia

Publication . Futema, Marta; Bourbon, Mafalda; Williams, Maggie; Humphries, Steve E.

Mutations in any of three genes (LDLR, APOB and PCSK9) are known to cause autosomal dominant FH, but a mutation can be found in only ∼40% of patients with a clinical diagnosis of FH. In the remainder, a polygenic aetiology may be the cause of the phenotype, due to the co-inheritance of common LDL-C raising variants. In 2013, we reported the development of a 12-SNP LDL-C "SNP-Score" based on common variants identified as LDL-C raising from genome wide association consortium studies, and have confirmed the validity of this score in samples of no-mutation FH adults and children from more than six countries with European-Caucasian populations. In more than 80% of those with a clinical diagnosis of FH but with no detectable mutation in LDLR/APOB/PCSK9, the polygenic explanation is the most likely for their hypercholesterolaemia. Those with a low score (in the bottom two deciles) may have a mutation in a novel gene, and further research including whole exome or whole genome sequencing is warranted. Only in families where the index case has a monogenic cause should cascade testing be carried out, using DNA tests for an unambiguous identification of affected relatives. The clinical utility of the polygenic explanation is that it supports a more conservative (less aggressive) treatment care pathway for those with no mutation. The ability to distinguish those with a clinical diagnosis of FH who have a monogenic or a polygenic cause of their hypercholesterolaemia is a paradigm example of the use of genomic information to inform Precision Medicine using lipid lowering agents with different efficacy and costs.

2018-10Journal article

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