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Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells

ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that targets for degradation faulty mRNAs with premature termination codons as well as many physiological mRNAs encoding full-length proteins. Consequently, NMD functions in both, quality control and post-transcriptional regulation of gene expression, and it has been implicated in the modulation of cancer progression. To investigate the role of NMD in cancer, we knocked out SMG7 in the HT1080 human fibrosarcoma cell line. SMG7 is involved in deadenylation-coupled exonucleolytic mRNA decay, one of the two main degradation pathways in mammalian NMD. Genome-wide proteomic and transcriptomic analyses confirmed that NMD is severely compromised in these SMG7-knockout HT1080 cells. We compared the oncogenic properties between the parental, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. In parallel, we tested the effect of a drug inhibiting the NMD factor SMG1 on the HT1080 cells to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays as well as a mouse xenograft tumor model, we show that the oncogenic properties of the parental HT1080 cells areseverely compromised when NMD is inhibited. Molecular pathway analysis revealed a strong reduction of the matrix metalloprotease 9 (MMP9) gene expression in NMD-suppressed cells. Since MMP9 expression promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation in NMD-suppressed cells explains, at least partially, their reduced tumorigenicity. Collectively, our findings emphasize the therapeutic potential of NMD inhibition for the treatment ofcertain types of cancer.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Manfred Heller

LAB HEAD: Oliver Mühlemann

PROVIDER: PXD041853 | Pride | 2023-09-19

REPOSITORIES: Pride

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Project description:Background The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA pro-cessing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oli-go(dT) beads and the RNA binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. Results Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seed-lings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5´untranslated region. The efficiency of RNA capture was greatly enhanced by the use of locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. In total, we identified more than 300 proteins interacting with the AtGRP7 RNA. These were benchmarked against proteins pulled down by AtGRP7 in vitro transcripts from nuclear lysates. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation. The circadian expression of the AtGRP7 transcript was slightly changed in an alba loss-of-function mutant compared to wild-type, demonstrating the functional relevance of the interaction. Conclusion We adapted specific RNP capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up-scaling of the protocol should be applicable for less abundant transcripts. Continuous improvements of mass spectrometry sensitivity will also help to increase the range of proteins that can be identified.

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	20170323_S1_SN_i16.raw	Raw
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Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells

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