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Acetylation of cytidines by NAT10 enhances HIV-1 replication through stabilization of viral RNA

ABSTRACT: As obligate parasites, viruses need to navigate a variety of cellular regulatory systems while infecting and replicating in the host cell. Post-transcriptional modifications have recently emerged as an important layer of regulation of viral RNA function. For example, our lab and others have shown that the RNA modification N6-methyladenosine (m6A) can enhance the replication of multiple viruses in cis, including Human Immunodeficiency virus 1 (HIV-1), Influenza A virus, SV40 and Kaposi's sarcoma-associated herpesvirus (KSHV). Recent reports have revealed the presence of another RNA modification, N4-acetylcytidine (ac4C) on cellular mRNAs and have shown that ac4C can enhance mRNA stability and translation. Here, we demonstrate that ac4C is present at multiple sites on HIV-1 mRNAs and on the viral genomic RNA. Through ac4C RNA immunoprecipitation (RNA-IP) and RNA-seq, we found ac4C on HIV-1 mRNAs as well as the virion genomic RNA, with ac4C sites in the coding regions of the pol, env, nef genes, and the trans-activation response (TAR) hairpin. Phenotypically, we observe that increasing the expression level of the ac4C acetyltransferase NAT10 leads to an increase in viral replication that is dependent on the RNA binding and enzymatic domains of NAT10. Moreover, both CRISPR-depletion of NAT10 (ΔNAT10) and treatment with the small molecule NAT10 inhibitor Remodelin, diminishes HIV-1 replication in T cells. Lastly, silent mutations introduced to prevent ac4C deposition on the viral genome indeed diminished HIV-1 replication. Our data suggest that HIV-1 has evolved to incorporate ac4C in essential viral gene coding regions and regulatory RNA structures, and that NAT10-dependent ac4C addition enhances HIV-1 replication.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142490 | GEO | 2020/06/11

REPOSITORIES: GEO

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Project description:Generation of the "epitranscriptome through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences. Ablation of NAT10 reduced ac4C detection at the mapped mRNA sites and was globally associated with target mRNA down-regulation. Analysis of mRNA half-lives revealed a NAT10-dependent increase in stability in the cohort of acetylated mRNAs. mRNA acetylation was further demonstrated to enhance substrate translation in vitro and in vivo. Codon content analysis within ac4C peaks uncovered a biased representation of cytidine within wobble sites that was empirically determined to influence mRNA decoding efficiency. These findings expand the repertoire of mRNA modifications to include an acetylated residue and establish a role for ac4C in the regulation of mRNA translation. Generation of the “epitranscriptome” through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here we describe N4-acetylcytidine (ac4C) as a novel mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were distributed across target mRNAs with the majority of peaks occurring within coding regions. Depletion of ac4C through NAT10 ablation revealed a relationship to gene expression wherein loss of ac4C was globally associated with transcript downregulation. The presence of ac4C within coding sequences was associated with elevated ribosome density and enhanced translation, as assessed in vivo and in vitro. In addition to expanding the repertoire of mRNA modifications to include an acetylated residue, these findings highlight a role for ac4C in the control of mRNA metabolism at the level of translation.

Project description:Background: Heart failure (HF), characterized by cardiac remodeling, is associated with abnormal epigenetic processes and aberrant gene expression. Here, we aimed to elucidate the effects and mechanisms of N-acetyltransferase 10 (NAT10)-mediated N4?acetylcytidine (ac4C) acetylation during cardiac remodeling. Methods: NAT10 and ac4C expression were detected in both human and mouse subjects with cardiac remodeling through multiple assays. Subsequently, acetylated RNA immunoprecipitation and sequencing (acRIP-seq), thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), and ribosome sequencing (Ribo-seq) were employed to elucidate the role of ac4C-modified post-transcriptional regulation in cardiac remodeling. Additionally, functional experiments involving the overexpression or knockdown of NAT10 were conducted in mice models challenged with Ang II and transverse aortic constriction (TAC). Results: NAT10 expression and RNA ac4C levels were increased in in vitro and in vivo cardiac remodeling models, as well as in patients with cardiac hypertrophy. Silencing and inhibiting NAT10 attenuated Ang II-induced cardiomyocyte hypertrophy and cardio-fibroblast activation. Next-generation sequencing revealed ac4C changes in both mice and humans with cardiac hypertrophy were associated with changes in global mRNA abundance, stability and translation efficiency. Mechanistically, NAT10 could enhance the stability and translation efficiency of CD47 and ROCK2 transcripts by upregulating their mRNA ac4C modification, thereby resulting in an increase in their protein expression during cardiac remodeling. Furthermore, the administration of Remodelin, a NAT10 inhibitor, has been shown to prevent cardiac functional impairments in mice subjected to TAC by suppressing cardiac fibrosis, hypertrophy, and inflammatory responses, while also regulating the expression levels of CD47 and ROCK2. Conclusions: Therefore, our data suggest that modulating epitranscriptomic processes, such as ac4C acetylation through NAT10, may be a promising therapeutic target against cardiac remodeling.

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Acetylation of cytidines by NAT10 enhances HIV-1 replication through stabilization of viral RNA

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