Percorrer por autor "Morgado, Ana"
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- Assessing the involvement of Dis3L1 in mammalian quality control pathwaysPublication . Reis, Filipa Pereira; Teixeira, Alexandre; Cruz, David; Morgado, Ana; Arraiano, Cecília Maria; Romão, LuísaThe exosome is an evolutionarily conserved protein complex that is involved in all aspects of RNA metabolism, namely, RNA decay, processing and quality control. The only catalytic subunit of the core exosome is a 3´end exoribonuclease from the RNase II family of enzymes. In humans, two different homologues of this protein were identified, Dis3 and Dis3L1. While Dis3 mainly localizes in the nucleoplasm and has endonucleolytic activity, Dis3L1 is strictly cytoplasmic and has no endonucleolytic activity. The rapid decay of aberrant transcripts is not completely understood, but it is known that involves both 5’ to 3’ and 3’ to 5’ degradation. Despite that it localizes in the same compartment where NMD generally occurs, nothing is known about the role of Dis3L1 in quality control processes. In this work, we assessed the involvement of Dis3L1 in the 3’ to 5’ degradation of reporter human b-globin transcripts with premature termination and nonstop codons.
- Unspliced Precursors of NMD-Sensitive β-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid CellsPublication . Morgado, Ana; Almeida, Fátima; Silva, Ana Luísa; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs). Mammalian NMD depends on both splicing and translation, and requires recognition of the premature stop codon by the cytoplasmic ribosomes. Surprisingly, some published data have suggested that nonsense codons may also affect the nuclear metabolism of the nonsense-mutated transcripts. To determine if nonsense codons could influence nuclear events, we have directly assessed the steady-state levels of the unspliced transcripts of wild-type and PTC-containing human β-globin genes stably transfected in mouse erythroleukemia (MEL) cells, after erythroid differentiation induction, or in HeLa cells. Our analyses by ribonuclease protection assays and reverse transcription-coupled quantitative PCR show that β-globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA. On the contrary, in HeLa cells, human β-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Functional analyses of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control. Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected. This set of data highlights potential nuclear pathways that might be promoter- and/or cell line-specific, which recognize the NMD-sensitive transcripts as abnormal. These specialized nuclear pathway(s) may be superimposed on the general NMD mechanism.