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An epitranscriptomic switch at the 5´-UTR controls genome selection during HIV-1 genomic RNA packaging

ABSTRACT: During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that an epitranscriptomic switch involving the demethylation of adenosine residues present within HIV-1 5´-UTR regulates full-length RNA packaging. We further identified two conserved adenosines within the 5’-UTR that have a crucial structural and functional role and that are modulated by N6-methylation. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promotes the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus contributing to full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.

ORGANISM(S): Homo sapiens HIV-1 vector pNL4-3

PROVIDER: GSE130687 | GEO | 2021/01/02

REPOSITORIES: GEO

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Analysis of lentiviral vector splicing events with nanopore sequencing

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FTO demethyltion of N6-methyladenosine depends on the RRACH motif [CLIP-seq]

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FTO Regulates Activation of Rho GTPases Signaling as N6-methyladenosine RNA Demethylase and an Rhotekin Partner Protein

Project description:Reversible RNA modification of N6-methyladenosine (m6A) plays a critical role in post-transcriptional gene regulation1-13. Although the fat mass and obesity-associated protein (FTO) has been previously shown to function as an m6A demethylase in nuclear RNA1,14, its exact function in disease pathogenesis remains a mystery. Here, we demonstrate that FTO suppresses the activation of Rho GTPase signaling via both its demethylation activity and specific interaction with Rho effector, Rhotekin (RTKN)15. The knockdown of FTO activates RhoA and RhoC, induces stress fibres, and accelerates cell migration. Endogenous RTKN is highly expressed in certain cancer and cancer-derived cell lines16,17 with overexpression of RTKN leading to moderate activation of Rho18. We further found that overexpression of RTKN blocks nuclear import of FTO and traps FTO in the cytoplasm to mediate m6A demethylation of cytosolic mRNA, thereby changing gene expression by preventing m6A-dependent mRNA decay and translation. Our results illustrate how FTO represses Rho activation through m6A demethylation and direct interaction with RTKN, and shed new light on FTO-dependent post-transcriptional gene regulation in RTKN overexpressed cancers, which may provide a new direction for developing anti-cancer therapies.

2018-12-31 | GSE83540 | GEO

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