DGH - Dissertações de mestrado
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- mRNA metabolism: nonsense-mediated mRNA decay as a tool for gene therapy and the role of human DIS3L2 in transcript degradationPublication . Asper, Gerson; Romão, Luísa; Dias, DeodáliaEukaryotic cells have developed elaborate mechanisms of mRNA quality control that secure gene expression fidelity through the detection and degradation of abnormal transcripts. NMD (nonsense-mediated mRNA decay), which detects and degrades transcripts containing premature translation termination codons (PTCs), and NSD (nonstop mRNA decay), that detects and degrades transcripts without in-frame stop codons, are just two examples. Nonsense-mediated mRNA decay (NMD) in particular, is a conserved surveillance system in all eukaryotic cells and is also the most extensively studied. PTC-containing mRNAs could, without NMD, give rise to C-terminally truncated proteins toxic for the cell. The physiological importance of NMD is further manifested by the fact that about one third of genetic disease-associated mutations generate PTCs. Recently, some studies have shown that aminoglycosides, low molecular weight compounds, and non-aminoglycosides can suppress PTCs in cystic fibrosis, Duchenne’s muscular dystrophy others, as a novel therapeutic approach, suppression therapy, which uses these compounds to induce recoding of a PTC into a sense codon. It is unclear whether β-thalassaemia would also be responsive to suppression therapy. Some recent studies show positive results for the compound PTC124 in suppressing nonsense mutations in the CFTR gene and others; also preliminary results obtained in our lab have shown that the aminoglycoside G418 can suppress a nonsense mutation at codon 39 of the human β-globin mRNA, although at low levels in cultured erythroid cells. As a first part of this work, we decided to investigate if suppression therapy can restore enough β-globin protein to correct the disease manifestations of β-thalassaemia. We intended to test whether G418 and/or PTC124 were able to induce efficient levels of suppression in a dose-dependent manner in HeLa cells transfected with plasmids containing the human β-globin wild type gene (βWT) or the other variants carrying a nonsense mutation at codon 15 (β15) or 39 (β39). However, we weren’t successful in this approach due to difficulties in gene cloning. The next step for RNAs targeted by NMD or NSD, as well as normal transcripts, which don’t accumulate indefinitely, is degradation. Generally the exosome complex, a multi-subunit ribonuclease complex, is responsible for the 3’-5’ degradation of every type of RNA in the cell, with its main catalytic component being either Dis3 or Dis3L1 in humans. However, another ribonuclease has been identified: DIS3L2. This protein is thought to be a cytoplasmic exosome-independent 3’-5’ ribonuclease, with special affinity for urydilated transcripts. Nonetheless, not much else is known for certain about its activity, especially in humans, including if it is coupled to NMD or NSD mRNA degradation. As a consequence, we intended to evaluate hDIS3L2’s possible involvement in mRNA degradation pathways, by performing knockdown of hDIS3L2, with siRNAs (small-interfering RNAs) in HeLa cells transfected with plasmids containing the human β-globin wild type gene (βWT), the variants carrying a nonsense mutation at codon 15 (β15), 26 (β 26) or 39 (β39), and also a variant lacking an in-frame stop codon (nonstop) (βNS). We then evaluated the human β-globin mRNA levels, as well as HFE’s, which is an NMD natural target. Our results show that human DIS3L2 is involved in NMD, NSD and possibly normal transcript degradation (mRNA turnover).