The involvement od DIS3L2 in nonsense-mediated mRNA decay and its functional networks in colorectal cancer

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The involvement of DIS3L2 in nonsense-mediated mRNA decay and its functional networks in cancer

Publication . García-Moreno, Juan F.; Romão, Luísa; Matos, Paulo

DIS3L2, member of the highly conserved RNase II family, is a 3’-5’ exoribonuclease that degrades different types of RNAs in an exosome-independent manner, including mRNAs and several types of non-coding RNAs. DIS3L2-mediated decay operates in tight cooperation with the terminal uridylyl transferases 4 and 7 (TUT4/7), which uridylate specific mRNAs that are subsequently recognized and degraded by DIS3L2. Recently, it was discovered that these three factors are involved in the nonsense-mediated mRNA decay (NMD) pathway. NMD is a surveillance pathway that selectively degrades mRNAs harboring premature translation-termination codons (PTCs) located more than 50-55 nucleotides upstream of an exon-exon junction, thus protecting the cell from the expression of truncated proteins. Furthermore, NMD controls the expression levels of normal and fully functional mRNAs, becoming an important mechanism of gene expression regulation in the cell. However, the mechanism by which DIS3L2 and uridylation take place in the NMD pathway remains poorly characterized. On the other hand, there is some evidence that dysregulated activity of DIS3L2 may contribute to cancer development. This ribonuclease has been implicated in cancer-related cellular processes such as cell proliferation and apoptosis, and mutations in DIS3L2 gene lead to deregulation of cell cycle genes inducing chromosomal instability. In this PhD project, we aimed to characterize the role of DIS3L2 in cancer and to dissect the mechanism by which DIS3L2 and uridylation are involved in the NMD pathway. By analyzing publicly available RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in colorectal cancer (CRC) tissues versus normal colonic samples as well as worse prognosis for CRC patients with high DIS3L2 expression. Moreover, our RNA deep-sequencing analysis showed that knockdown (KD) of DIS3L2 results in a significant transcriptomic disturbance in SW480 CRC cells. In addition, gene ontology (GO) analysis of significant upregulated genes exhibited enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to investigate which specific hallmarks of cancer are differentially regulated by DIS3L2. Four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) with distinct mutational background and oncogenicity were used to test the role of DIS3L2 in this cancer type. Our results indicate that lack of DIS3L2 impairs cell viability of highly oncogenic SW480 and HCT116 CRC cells, but has little or no impact in the more differentiated Caco-2 and HT 29 cells. Interestingly, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated upon DIS3L2 depletion, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, we demonstrate that DIS3L2 KD leads to the disruption of metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our bioinformatic analysis corroborate the predictive value of specific NMD-eliciting genomic features characterized in previous studies, such as a high GC content in the 3’UTR, the presence of 3’UTR introns or a higher incidence of uORFs. Interestingly, the 3’UTRs of NMD-targeted mRNAs degraded by DIS3L2 tend to have a high GC content and to be devoid of 3’UTR introns when compared to 3’UTRs of transcripts showing resistance to NMD and DIS3L2-mediated degradation. Notably, gene expression assessment in a system of constructs containing different genomic regions of the NMD/DIS3L2 target GADD45A unveiled that its 3’UTR enhances susceptibility to NMD and contributes for DIS3L2 target recognition. Furthermore, we discovered that interruption of the mRNA closed-loop configuration does not sensitize NMD targets to degradation by DIS3L2. Curiously, we found that DIS3L2-mediated degradation seems to be more efficient in circularized NMD targets. Throughout this work, several results have shown that lack of uridylation along with DIS3L2 mitigates the impact observed upon single depletion of this ribonuclease. In an attempt to identify additional factors contributing to this phenomenon, we demonstrate that neither of the canonical cytoplasmic exoribonucleases DIS3, DIS3L1 nor XRN1 is responsible for such compensatory mechanism. Altogether, the work here presented unveils a novel oncogenic role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells. Moreover, this work shed light on the mechanism by which DIS3L2 is involved in NMD, highlighting the biological relevance of the 3’UTR on DIS3L2 target recognition. Taking into account that the NMD pathway regulates important biological processes in the pathophysiology of cancer, understanding the mechanism by which DIS3L2 regulates specific NMD targets may provide vital information for the development of new therapeutic strategies.

2023-12Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Número da atribuição

PD/BD/142898/2018