Browsing by Author "Viegas, Sandra C."
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- 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.
- 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.
- 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.
- The Implication of mRNA degradation disorders on human dISease: focus on DIS3 and DIS3-Like enzymesPublication . Saramago, Margarida; da Costa, Paulo J.; Viegas, Sandra C.; Arraiano, Cecília M.RNA degradation is considered a critical posttranscriptional regulatory checkpoint, maintaining the correct functioning of organisms. When a specific RNA transcript is no longer required in the cell, it is signaled for degradation through a number of highly regulated steps. Ribonucleases (or simply RNases) are key enzymes involved in the control of RNA stability. These enzymes can perform the RNA degradation alone or cooperate with other proteins in RNA degradation complexes. Important findings over the last years have shed light into eukaryotic RNA degradation by members of the RNase II/RNB family of enzymes. DIS3 enzyme belongs to this family and represents one of the catalytic subunits of the multiprotein complex exosome. This RNase has a diverse range of functions, mainly within nuclear RNA metabolism. Humans encode two other DIS3-like enzymes: DIS3L (DIS3L1) and DIS3L2. DIS3L1 also acts in association with the exosome but is strictly cytoplasmic. In contrast, DIS3L2 acts independently of the exosome and shows a distinctive preference for uridylated RNAs. These enzymes have been shown to be involved in important cellular processes, such as mitotic control, and associated with human disorders like cancer. This review shows how the impairment of function of each of these enzymes is implicated in human disease.
- Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancerPublication . Costa, Paulo J. da; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe final step of eukaryotic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by DIS3, DIS3L1 (the catalytic subunits of the exosome) and/or DIS3L2 (exosome-independent). 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. In this work, 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. Each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several endogenous targets was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in any known mRNA decay mechanisms such as nonsense-mediated decay (NMD). However, we do not know yet what defines such target preference. In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1 or UPF1 (which has a central role in NMD) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data. Presently, we are exploring the mechanism through which DIS3L2 is involved in NMD and how it modulates the expression of NMD targets.