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- Dissecting the DIS3L2 target-specificity of transcripts committed to nonsense-mediated decay in human cellsPublication . Garcia-Moreno, Juan F.; Carvalho, Miguel P.; Lacerda, Rafaela; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates mRNAs harboring premature termination codons (PTCs) and regulates the expression of certain physiological transcripts. The 3’-to-5’ exoribonuclease DIS3L2 degrades different RNAs independently of the RNA exosome, following uridylation at the 3' end by the terminal uridylyl transferases TUT4 and TUT7. We and others have shown that DIS3L2 is involved in NMD in an uridylation-dependent manner, being its function in NMD target-specific (Kurosaki et al. 2018; da Costa et al. 2019). Now, we aim to characterize the mechanisms involved in DIS3L2/NMD-target specificity. We used our RNA-seq data already obtained and validated and compared the transcripts upregulated upon DIS3L2 knockdown (REF) with a validated NMD-target set (Colombo et al., 2017). We observed that about 7% of DIS3L2-sensitive transcripts overlap with known NMD-targets. Considering the different groups of transcripts, we then analyzed specific features that make some NMD-targets sensitive to DIS3L2 (so called DIS3L2/NMD-targets; group 1), versus the remaining NMD-targets (DIS3L2-resistant NMD-targets; group 2), the remaining DIS3L2-sensitive targets (group 3), or the remaining transcriptome (DIS3L2-resistant NMD-targets plus NMD-resistant transcriptome; group 4). We assessed the following genomic features: 5’ and 3’ untranslated region (UTR) lengths, 3’UTR GC-, AU-, G-, C-, A-, and U-contents, presence of 5’UTR upstream open reading frames (uORFs), and 3’UTR introns. Elevated G-, C-, and GC-contents in the 3’UTRs were the most consistent features distinguishing DIS3L2/NMD-targets from the group 4. Comparison between group 1 and 2, and 1 and 3 was not significant. To better characterize the importance of each transcript portion, we are also analyzing hybrid constructs combining regions of the DIS3L2/NMD-resistant human β-globin (HBB) gene and the DIS3L2/NMD-sensitive GADD45A gene expressed in DIS3L2 depleted cultured cells.
- The potential function of alternative translation initiation of Argonaute 1 in cancerPublication . Vieira da Silva, Verónica; Lacerda, Rafaela; Romão, LuísaTranslation is one of the most regulated and energy-consuming cellular processes crucial for proper cell function. Translation is initiated by the canonical cap-dependent mechanism. However, under stress conditions, the initiation of canonical translation is inhibited, which allows the translation of specific proteins via alternative mechanisms. This project aims to understand the biological relevance of alternative protein synthesis mechanisms in Argonaute 1 (AGO1) expression. The AGO1 protein is involved in microRNA regulation, gene expression modulation and inhibition. AGO1 is also involved in the regulation of gene expression by RNA interference (RNAi), and its deregulation can lead to the activation of oncogenes or the suppression of tumor suppressor genes, contributing to the development and progression of cancer. Our work has shown that AGO1 mRNA can be translated through a cap-independent initiation mechanism. An upstream open reading frame (uORF) has also been identified in its 5’ untranslated region (5’UTR), which may play a role in the initiation of AGO1 translation. The results showed that the 5’UTR of human AGO1 supports a cap-independent mechanism of translation initiation, which is maintained under stress conditions. However, our analyses did not provide conclusive evidence for a regulatory role of the uORF in this initiation process. To this end, the 5’UTR of human AGO1 was cloned into a bicistronic vector containing Renilla (RLuc) and Firefly luciferase (FLuc), with FLuc positioned downstream of the 5’UTR. Luminometry assays will be used to evaluate the relative FLuc/RLuc translation efficiency under the control of the AGO1 5’UTR. The same approach will be used with monocistronistic reporter vectors, contaning only FLuc. This project aims to understand how these alternative mechanisms of mRNA translation initiation can influence AGO1 expression and help explain their potential roles in certain pathologies and cancer progression, such as colorectal cancer.
- Upstream Open Reading Frames Regulate PERK Translation InitiationPublication . Fernandes, Rafael; Silvestre, Samuel; Ponte, João; Lacerda, Rafaela; Romão, LuísaIntroduction: Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS).
- Workforce Skills And Organisation: Perspectives From Educators And Health ProfessionalsPublication . Vicente, Astrid MouraThe presentation focuses on workforce skills and organizational perspectives from educators and health professionals within the scope of the Beyond 1 Million Genomes Plus (B1MGplus) project, which aims to create a European cross-border network of genomic and clinical data to improve healthcare outcomes.