Browsing by Issue Date, starting with "2019-06-11"
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- Translation of the human ABCE1 transcript is regulated by upstream open reading framesPublication . Silva, Joana; Romão, LuísaShort upstream open reading frames (uORFs) are cis-acting elements located within the 5'-leader sequence of transcripts and are defined by an initiation codon in-frame with a termination codon located upstream or downstream of its main ORF (mORF) initiation codon. Recent genome-wide ribosome profiling (Ribo-seq) studies have confirmed the widespread presence of uORFs and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream mORF by triggering mRNA decay or by regulating translation. Based on 5’-leader sequence ribosome occupancy profiles from Ribo-seq analysis in HCT116 cells, we studied the role of 6 non-AUG and 5 AUG uORFs present in the human ABCE1 mRNA. Using a set of reporter genes expressed in HCT116 cells and luminometry assays, we have observed that there are three AUG uORFs acting in a fail-safe manner to inhibit translation from the main AUG, being this repression immune to eIF2 phosphorylation. Functional aspects and implications of this regulatory mechanism to cell physiology will be discussed.
- 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 PERK mRNA: an unexpected non-NMD-targetPublication . Fernandes, Rafael; Romão, LuísaNonsense-mediated mRNA decay (NMD) was firstly described as a surveillance pathway that recognizes and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs) resulted from mutations or errors in RNA processing. Many transcriptome-wide studies demonstrated that NMD also targets mRNAs transcribed from a large subset of wild-type genes, thus arising as a post-transcriptional regulatory mechanism of gene expression. Hence, NMD contributes to the regulation of many essential biological processes, including stress responses. Recent reports revealed that NMD is capable of regulating the Unfolded Protein Response (UPR) that is induced in conditions of endoplasmic reticulum (ER) stress. This is achieved, at least in part, by the degradation of the IRE1α mRNA, a natural NMD-target and one of the three primary factors of the UPR. The other two factors are ATF6 and PERK, and there are evidences suggesting that the PERK-branch can also be involved in the NMD-mediated regulation of the UPR. In this work, we intended to test if the PERK mRNA is a natural NMD-target and, if so, determine the role and relevance of NMD to the PERK-mediated response during the UPR. By using 5’/3’ rapid amplification of cDNA ends (RACE) we have confirmed the sequences and lengths of the annotated 5’ and 3’ untranslated regions (UTRs) of the PERK mRNA. This allowed us to confirm the presence of two possible NMD-inducing features: upstream open reading frames (uORFs) in the 5’UTR and a long 3’UTR (~100bp). To test if PERK mRNA is a direct NMD-target, we have assessed its mRNA levels and stability in conditions of impaired NMD by UPF1 (NMD central factor) siRNA-mediated knockdown in HeLa cells. Surprisingly, the UPF1 knockdown did not induce the upregulation of PERK mRNA and neither stabilize it, suggesting that PERK mRNA is not a natural NMD-target. Our data suggests that NMD does not regulate the UPR through degradation of the PERK mRNA as it does with the IRE1α mRNA. However, we cannot rule out the hypothesis of NMD acting in the PERK-branch, for instance, by degrading downstream targets of PERK.