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- Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancerPublication . Costa, Paulo; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe final step of cytoplasmic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by the exosome and DIS3L2. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome. In humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. Important findings over the last years have shed a new light onto the mechanistic details of RNA degradation by these exoribonucleases. In addition, it has been shown that they are involved in growth, mitotic control and important human diseases, including cancer. For example, DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins (1). In another study, DIS3 was found to be highly expressed in colorectal cancer (CRC), suggesting an oncogenic function (2). A major challenge in systems biology is to reveal the cellular networks that give rise to specific phenotypes (3). In this project, we aim to analyze how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, and how their functional interactions modulate the transcriptional reprogramming of colorectal cancer cells. In order to unveil the role of these exoribonucleases in general mRNA decay, and/or in cytoplasmic mRNA surveillance mechanisms, such as nonstop- and nonsense-mediated decay (NSD and NMD), we performed their knockdown and measured the mRNA levels of various reporter transcripts (endogenous and exogenous), with emphasis in natural NMD targets. Our results show that DIS3 and DIS3L1 seem to be involved in the normal mRNA turnover, as well as in the NSD and NMD mechanisms. However, some natural NMD targets are resistant to these nucleases. On the other hand, DIS3L2 is not involved in the normal mRNA turnover or in NSD, being specifically involved in the degradation of some NMD targets. Presently, we are interested in identifying the transcript features implicated in the decision-making process of DIS3L2-mediated decay of natural NMD targets, as well as the corresponding mechanism. With this purpose, we performed a bioinformatics analysis of available transcriptomic data from DIS3, DIS3L1, DIS3L2+XRN1, XRN1, or UPF1 (a central player in NMD) knockdown experiments and identified transcripts differentially expressed in each condition. Results show some, but not total, redundancy between the upregulated transcripts, and this supports our experimental data.
- Regulation of nonsense-mediated mRNA decay (NMD) and the transcriptome: implications for physiology and myocardial infarctionPublication . Fernandes, Rafael; Bourbon, Mafalda; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs) that would otherwise lead to the production of potentially harmful truncated proteins (1). Recent studies demonstrated that NMD also targets physiologic mRNAs transcribed from a large subset of wild-type genes, being responsible for the regulation of up to 10% of the mammalian transcriptome (2,3). This raises the possibility that NMD itself is under regulatory control. Indeed, recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including NMD itself (4). Cellular stress, such as endoplasmic reticulum (ER) stress, hypoxia, reactive oxygen species, and nutrient deprivation also modulates the magnitude of NMD by mechanisms that are beginning to be understood (5). For example, the activation of kinases, as part of the cell-stress corrective pathways, induces the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), reducing protein translation and thus impairing NMD activity (6,7). There is currently great interest in decoding the mechanisms that couple stress signaling to human pathology. Only recently has ER stress been considered a potential contributor to cardiac and vascular diseases (8). Myocardial infarction is a pathological state that occurs during ischemia, where there is nutrient and oxygen deprivation in the heart, causing aggregation of proteins in the ER. This aggregation triggers ER stress and the three arms of the unfolded protein response (UPR), to mitigate or eliminate the stress (8). Given that NMD can respond to ER stress (6), here we aim to study 1) how NMD is regulated in cardiomyocytes under stress conditions, and 2) what is the influence of NMD on the transcriptome of cardiomyocytes and how it is involved in the cell-stress corrective strategies. So far, we have built a database that contains around 149 transcripts which are natural NMD-targets and are dysregulated under stress conditions, based on data from transcriptomic and UPF1-silencing studies. Using bioinformatics and gene ontology analysis we have classified the transcripts by biological function, and we intend to choose a few molecular targets for further studies in cardiomyocytes cultured cells in order to accomplish the objectives proposed.