Browsing by Author "Saramago, Margarida"
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- An unexpected role for DIS3L2 over human NMD targetsPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; 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. 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, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however nothing 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 are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD targets are in fact direct DIS3L2 substrates. We also observed that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation in NMD.
- An unexpected role for DIS3L2 over human NMD targetsPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; 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. In another study, DIS3 was found to be highly expressed in colorectal cancer (CRC), suggesting an oncogenic function. A major challenge in systems biology is to reveal the cellular networks that give rise to specific phenotypes. 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.
- Behind the curtain: unveiling DIS3L2 role in NMD and human cancerPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance mechanism that targets and degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins potentially harmful for the cells. In addition to this, several studies have shown that NMD regulates the levels of many physiological mRNAs that encode full-length proteins. Nevertheless, NMD is inhibited in tumor microenvironment and (de)regulates oncogenes and tumor suppressors in several types of cancer. In humans, the mRNA degradation pathways involve exonuclease proteins, such as the DIS3-like protein family (DIS3, DIS3L1 and DIS3L2); however, it is not known whether these proteins are involved in NMD. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown, by RNA interference, in HeLa cells and measured, by RT-qPCR, the mRNA levels and half-lives of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicate that these NMD targets are in fact direct DIS3L2 substrates. By performing DIS3L2, TUT4 and TUT7 triple knockdown, we also observed that DIS3L2-mediated decay depends on the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4) activity. Among the NMD targets regulated by DIS3L2, we highlight GADD45A. GADD45A is involved in cell cycle arrest, DNA damage response and apoptotic process. Furthermore, GADD45A deregulation is associated with several types of cancer, such as, esophageal, lung, bladder and pancreatic. Together, our findings establish the role of DIS3L2 and uridylation in NMD and in the regulation of oncogenes and tumor suppressor gene expression. These results might be highly relevant for the advance in diagnosis, prognosis and treatment of many human cancers.
- A Comparative Overview of the Role of Human Ribonucleases in Nonsense-Mediated mRNA DecayPublication . da Costa, Paulo J.; Menezes, Juliane; Guedes, Raquel; Reis, Filipa P.; Teixeira, Alexandre; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília M.; Romão, LuísaEukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.
- 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.
- How DIS3L2 meets NMD-targets: I’m really into “U”!Publication . da Costa, Paulo J.; Saramago, Margarida; Viegas, Sancra C.; Arraiano, Cecília M.; 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. Also, NMD regulates the levels of many physiological PTC-free 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 achieved by a process not yet completely understood that promotes the decay of the mRNAs in 5’-to-3’ and 3’-to-5’ by the XRN1 and exosome, respectively. 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 the Perlman syndrome-associated exoribonuclease DIS3L2. Conversely, to its counterparts, DIS3L2 activity is independent of the exosome. 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 DIS3L2 is involved in NMD-targets decay. Besides that, DIS3L2 acts directly on NMD-targets and interacts with the key NMD factor UPF1. We also show that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) 4 and 7, which adds non-templated uridines to the mRNAs 3’ end, marking these mRNAs for DIS3L2 degradation. Together, our findings establish a direct role of DIS3L2 in NMD in an uridylation-dependent manner.
- 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.
- Ribonucleases and nonsense-mediated decay (NMD): An unexpected role for DIS3L2 over human NMD targetsPublication . da Costa, Paulo J.; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília M.; Romão, LuísaBackground: The 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. 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, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however nothing 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 are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD targets are in fact direct DIS3L2 substrates. We also observed that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation in NMD.
- 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.
- The function of DIS3L2 in the mechanism of nonsense-mediated mRNA decayPublication . Garcia-Moreno, Juan; da Costa, Paulo J.; Menezes, Juliane; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília C.; 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 work, we show the DIS3L2 involvement in the NMD target regulation, by its dependent action on the NMD central player, UPF1. We also 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.