Browsing by Author "García-Moreno, Juan F."
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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.
- DIS3L2: Unveiling a New Player in Tumorigenesis, with a Key Role in Colorectal CancerPublication . García-Moreno, Juan F.; Matos, Paulo; Romão, LuísaDIS3L2 is a 3’-5’ exoribonuclease that recognizes and degrades uridylated transcripts in an exosome-independent manner and participates in several RNA degradation pathways, such as the nonsense-mediated mRNA decay, or the surveillance of aberrant structured non-coding RNAs. Although some studies have linked DIS3L2 to tumorigenesis and cancer-related processes, its exact role in the development and progression of cancer has remained unclear since the discovery of DIS3L2's ribonuclease activity a decade ago. While some authors have reported evidence of a tumor suppressor role for this exoribonuclease, other studies have shown DIS3L2 as a driver of tumorigenesis. Although differences in tissue type and methodologic approaches may somewhat account for the opposing findings, a recent study from our group further supports a pro-tumorigenic role for DIS3L2, this time in promoting colorectal cancer (CRC) progression. Indeed, proper DIS3L2 expression was proven essential to maintain key tumorigenic properties in CRC cells, including cell proliferation and invasion. Here, we summarize the current state of knowledge regarding the impact of DIS3L2 in cancer, namely in colorectal cancer. The collected data unveils DIS3L2 as a novel putative therapeutic target in cancer that warrants further investigation.
- Experimental supporting data on DIS3L2 over nonsense-mediated mRNA decay targets in human cellsPublication . da Costa, Paulo J.; Menezes, Juliane; Saramago, Margarida; García-Moreno, Juan F.; Santos, Hugo A.; Gama-Carvalho, Margarida; Arraiano, Cecília M.; Viegas, Sandra C.; Romão, LuísaIn this article, we present supportive data related to the research article “A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells” [1], where interpretation of the data presented here is available. Indeed, here we analyze the impact of the DIS3L2 exoribonuclease over nonsense-mediated mRNA decay (NMD)-targets. Specifically, we present data on: a) the expression of various reporter human β-globin mRNAs, monitored by Northern blot and RT-qPCR, before and after altering DIS3L2 levels in HeLa cells, and b) the gene expression levels of deregulated transcripts generated by re-analyzing publicly available data from UPF1-depleted HeLa cells that were further cross-referenced with a dataset of transcripts upregulated in DIS3L2-depleted cells. These analyses revealed that DIS3L2 regulates the levels of a subset of NMD-targets. These data can be valuable for researchers interested in the NMD mechanism.
- Mechanistic aspects of nonsense-mediated mRNA decay in human cellsPublication . Costa, Paulo J. da; Menezes, Juliane; Saramago, Margarida; García-Moreno, Juan F.; Santos, Hugo A.; Gama-Carvalho, Margarida; Arraiano, Cecília M.; Viegas, Sandra C; Romão, LuísaThe nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. NMD results from improper translation termination at stop codons, and thus, it is a cytoplasmic and translation-dependent process. In human cells, mRNA decay inherent to NMD involves an endonucleolytic cleavage near the stop codon and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD-targets. Our results show that some NMD-targets accumulate in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD-targets are direct DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, being DIS3L2-mediated decay dependent on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation over human NMD-targets.
- A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cellsPublication . Costa, Paulo J. da; Menezes, Juliane; Saramago, Margarida; García-Moreno, Juan F.; Santos, Hugo A.; Gama-Carvalho, Margarida; Arraiano, Cecília M.; Romão, LuísaThe nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-' and 3'-ends. This is done by a process not yet completely understood that recruits decapping and 5'-to-3' exonuclease activities, as well as deadenylating and 3'-to-5' exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.