Browsing by Issue Date, starting with "2018-07-02"
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- Gene expression regulation by upstream open reading frames in colorectal cancerPublication . Silva, Joana; Romão, LuísaColorectal cancer (CRC) has a high incidence and mortality rates worldwide. Its carcinogenesis process is characterized by a continuous accumulation of genetic alterations that changes the overall gene expression profiles. Those alterations have been studied by microarray and RNA sequencing that measure the abundance of mRNA but do not provide information on protein synthesis, a step closer to the end-point of gene expression. Ribosome profiling (RiboSeq) emerges to monitor in vivo translation by deep sequencing of ribosome-protected mRNA fragments (RPFs). This technique detects ribosomes outside of known protein-coding regions, identifying translation of upstream open reading frames (uORFs) within 5’ untranslated regions (5’UTRs). The aim of this work is to determine the role of specific uORFs in CRC tumorigenesis. For that, we looked for potential uORFs-containing targets based in the 5’UTR RPFs occupancy from RiboSeq data from different cancer cell lines already available. We chose ABCE1, PAIP2, eIF4G2 and eIF2A as uORFs-containing mRNAs. Gene ontology analyses revealed an important role in translational control for the proteins encoded by these transcripts. By semi-quantitative RT-PCR, ABCE1 transcript is shown down-regulated in HCT116 cells in comparison to the non-neoplasic colorectal cell line (NCM460). To analyze the role of such uORFs in translational regulation and their biological function at the level of cell viability and proliferation, and acquisition of CRC features, a reporter plasmid was constructed carrying the ABCE1 5’UTR fused to the Firefly luciferase (Fluc) ORF (pGL2-ABCE1). Each one of the five upstream AUGs in ABCE15’UTR was mutated to obtained constructs with non-functinal uORFs and only one functional uORF. HCT116 cells were transiently transfected with pGL2-ABCE1 or each one of the above mentioned constructs. Fluc expression and activity was assessed by Western blot and luminometry assays, respectively. Results show a decrease in the translational efficiency of Fluc by pGL2-ABCE1. Moreover, the construct carrying only the uORF3 functional exhibits a stronger repression efficacy compared to pGL2-ABCE1 and the other constructs.
- The interplay between nonsense-mediated mRNA decay (NMD) and the unfolded protein response (UPR) in myocardial infarctionPublication . Fernandes, Rafael; Bourbon, Mafalda; Romão, LuísaMany genome-wide studies pointed out translation as a major regulator of gene expression, being a key post-transcriptional mechanism by which cells rapidly change their gene expression pattern in response to diverse stimuli. There are several cis-acting elements that can be involved in the regulation of translation initiation, including upstream open reading frames (uORFs). A uORF is defined as a coding sequence that is located within the 5’ untranslated region (5’UTR) of a transcript, and it is typically considered an inhibitor of downstream translation initiation at the main ORF (mORF). This can be due to the recognition of the uORF start codon by the preinitiation complex. In this case, when the translating ribosome encounters the stop codon of the uORF, the translation machinery disassembles, a fact that can avoid translation of the mORF if the ribosome cannot reinitiate at the main start codon. ATF4, CHOP and GADD34 are stress-response proteins encoded by uORF-harboring transcripts with translation repression activity, which is responsible for maintaining a low expression of these proteins in normal conditions. However, when ER stress occurs, the unfolded protein response (UPR) is activated and eIF2α is phosphorylated by PERK. In these cases, the availability of the preinitiation complex is reduced, favoring translation of the mORFs. The stress-response proteins are therefore up regulated, triggering a cascade of events aiming stress resolution and cell survival. Given that many factors of the PERK-pathway of the UPR are regulated by uORFs, in this work we intended to determine if PERK is also regulated at the translational level. We have started by validating the annotated sequence of PERK 5’UTR using 5’RACE. Then, we have selected uORFs based on ribosome profiling data already available. To study the role of these uORFs in translational regulation of PERK, we have cloned its 5’UTR into a reporter plasmid, in frame with firefly luciferase ORF. Using site-directed mutagenesis, we have made constructs with mutated uORFs to evaluate their impact in translation efficiency. Our data, suggests that the uORFs have a repressive effect in mORF translation, and we are now dissecting the mechanisms that drive this regulation, in normal and ER stress conditions.
- 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.