Author(s): Morgado, Ana Sofia João
Date: 2013
Persistent ID: http://hdl.handle.net/10362/8798
Origin: Repositório Institucional da UNL
Subject(s): Nonsense-mediated mRNA decay (NMD); mRNA metabolism; Nuclear mRNA surveillance; Human β-globin pre-mRNA; Mouse erythroleukemia (MEL) cells
Dissertação para obtenção do Grau de Doutor em Biologia, Especialidade de Biologia Molecular
Nonsense-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. Therefore, we hypothesized that human β-globin transcripts sensitive to NMD could have a singular subcellular localization and processing state in mammalian cells nuclei. To determine if PTCs could influence nuclear events, we have established mouse erythroleukemia (MEL) cell lines stably transfected with wild-type or PTC-containing human β-globin genes. Subsequently, we analyzed the accumulation of NMD-competent β-globin transcripts versus wild-type counterparts using two different approaches: visualization of transcripts localization by fluorescence in situ hybridization (FISH); and quantification of pre-mRNA steady-state levels by ribonuclease protection assays (RPA) and reverse transcription-coupled quantitative polymerase chain reaction (RT-qPCR). FISH analysis shows that MEL cells stably expressing PTC-containing β-globin transcripts present a marked tendency to display an abnormal speckled-like pattern of localization in the nucleus. However, in addition to the presence of the PTC, other effectors may act on the β-globin transcripts localization, as some wild-type β-globin MEL cells presented this abnormal FISH phenotype as well. On the other hand, our analyses by RPA and RT-qPCR clearly 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. Conversely, in non-erythroid HeLa cells, human β-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Half-life analysis 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. In conclusion, our set of data highlights potential nuclear pathways that induce a selective downregulation of PTC-containing β-globin pre-mRNA in MEL cells, albeit not affecting their stability or splicing effectiveness. These specialized nuclear pathways, which may act in concert with the general NMD mechanism, might discriminate the NMD-sensitive transcripts as abnormal in a promoter- and/or cell line-specific manner, probably to obtain optimal NMD activity.
Fundação para a Ciência e Tecnologia - (SFRH/BD/31920/2006); financial support [Centro de Investigação em Genética Molecular Humana (CIGMH) and Center for Biodiversity, Functional and Integrative Genomics (BioFIG)]
