Browsing by Author "Pereira, Marcelo"
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- DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathwayPublication . García-Moreno, Juan F.; Lacerda, Rafaela; da Costa, Paulo J.; Pereira, Marcelo; Gama-Carvalho, Margarida; Matos, Paulo; Romão, LuísaDIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3’end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that lack depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.
- Functional networks of DIS3L2 in cancer and NMDPublication . García-Moreno, Juan; da Costa, Paulo J; Menezes, Juliane; Pereira, Marcelo; Gama-Carvalho, Margarida; Matos, Paulo; Romão, LuísaIn the flow of information from DNA to mRNA to proteins, mRNAs undergo a number of processing steps, since they are synthesized in the nucleus, until they are translated in the cytoplasm. Eukaryotic cells tightly control the fidelity of this process, via quality control pathways, among them, the nonsense-mediated mRNA decay (NMD). NMD recognizes and degrades mRNAs harboring premature translation-termination codons (PTCs), protecting the cell from potentially harmful truncated proteins. However, NMD can also regulate normal and fully functional mRNA levels, arising as a surveillance and a gene expression regulation pathway. A new branch of the NMD pathway is starting to be revealed, which is characterized by the involvement of the DIS3L2 3’ to 5’ exoribonuclease. This protein, has special relevance, given its exosome-independent action and its uridylation-mediated decay. In addition, mutations on this ribonuclease, induce deregulation of cell-cycle genes leading to a faster cell growth and decreased chromosome stability, while DIS3L2 downregulation enhances cancer stem cell properties. Several lines of evidence point to an oncogenic role of DIS3L2 and its mediated decay over a number of NMD targets, however further research is needed to unveil the mechanism by which this nuclease is involved in NMD and how it mediates cancer related processes. In this project, we aim to analyze how DIS3L2 and uridylation regulate the human transcriptome, in order to shed light on how this ribonuclease is related to NMD and how its deregulation contributes to tumorigenesis. For this purpose, high-throughput mRNA sequencing has been performed in the SW480 colorectal cancer cell line depleted of DIS3L2 or DIS3L2 plus terminal uridylyl transferases (TUTases), TUT4 and TUT7. Preliminary results show gene ontology enrichment in molecular functions and biological processes related with cancer.
- Networks of mRNA Processing and Alternative Splicing Regulation in Health and DiseasePublication . Jordan, Peter; Gonçalves, Vânia; Fernandes, Sara; Marques, Tânia; Pereira, Marcelo; Gama-Carvalho, MargaridamRNA processing events introduce an intricate layer of complexity into gene expression processes, supporting a tremendous level of diversification of the genome's coding and regulatory potential, particularly in vertebrate species. The recent development of massive parallel sequencing methods and their adaptation to the identification and quantification of different RNA species and the dynamics of mRNA metabolism and processing has generated an unprecedented view over the regulatory networks that are established at this level, which contribute to sustain developmental, tissue specific or disease specific gene expression programs. In this chapter, we provide an overview of the recent evolution of transcriptome profiling methods and the surprising insights that have emerged in recent years regarding distinct mRNA processing events - from the 5' end to the 3' end of the molecule.