Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability.
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ABSTRACT:
SUBMITTER: Tsai K
PROVIDER: S-EPMC7429276 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease.
| S-EPMC8054311 | biostudies-literature
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.
2018-11-07 | GSE102113 | GEO
Project description:BackgroundThe human immunodeficiency virus type 1 (HIV-1) structural protein Gag is necessary and sufficient to form viral particles. In addition to encoding the amino acid sequence for Gag, the underlying RNA sequence could encode cis-acting elements or nucleotide biases that are necessary for viral replication. Furthermore, RNA sequences that inhibit viral replication could be suppressed in gag. However, the functional relevance of RNA elements and nucleotide biases that promote or repress HIV-1 replication remain poorly understood.ResultsTo characterize if the RNA sequence in gag controls HIV-1 replication, the matrix (MA) region was codon modified, allowing the RNA sequence to be altered without affecting the protein sequence. Codon modification of nucleotides (nt) 22-261 or 22-378 in gag inhibited viral replication by decreasing genomic RNA (gRNA) abundance, gRNA stability, Gag expression, virion production and infectivity. Comparing the effect of these point mutations to deletions of the same region revealed that the mutations inhibited infectious virus production while the deletions did not. This demonstrated that codon modification introduced inhibitory sequences. There is a much lower than expected frequency of CpG dinucleotides in HIV-1 and codon modification introduced a substantial increase in CpG abundance. To determine if they are necessary for inhibition of HIV-1 replication, codons introducing CpG dinucleotides were mutated back to the wild type codon, which restored efficient Gag expression and infectious virion production. To determine if they are sufficient to inhibit viral replication, CpG dinucleotides were inserted into gag in the absence of other changes. The increased CpG dinucleotide content decreased HIV-1 infectivity and viral replication.ConclusionsThe HIV-1 RNA sequence contains low abundance of CpG dinucleotides. Increasing the abundance of CpG dinucleotides inhibits multiple steps of the viral life cycle, providing a functional explanation for why CpG dinucleotides are suppressed in HIV-1.
| S-EPMC5679385 | biostudies-literature
Project description:The human acetyltransferase NAT10 has recently been shown to catalyze formation of N4-acetylcytidine (ac4C), a minor nucleobase known to alter RNA structure and function. In order to better understand the role of RNA acetyltransferases in biology and disease, here we report the development and application of chemical methods to study ac4C. First, we demonstrate that ac4C can be conjugated to carrier proteins using optimized protocols. Next, we describe methods to access ac4C-containing RNAs, enabling the screening of anti-ac4C antibodies. Finally, we validate the specificity of an optimized ac4C affinity reagent in the context of cellular RNA by demonstrating its ability to accurately report on chemical deacetylation of ac4C. Overall, these studies provide a powerful new tool for studying ac4C in biological contexts, as well as new insights into the stability and half-life of this highly conserved RNA modification. More broadly, they demonstrate how chemical reactivity may be exploited to aid the development and validation of nucleobase-targeting affinity reagents designed to target the emerging epitranscriptome.
| S-EPMC7900898 | biostudies-literature
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 downregulation. 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.
| S-EPMC6295233 | biostudies-literature
Project description:Integration of HIV-1 into the human genome, which is catalyzed by the viral protein integrase (IN), preferentially occurs near transcriptionally active genes. Here we show that p300, a cellular acetyltransferase that regulates chromatin conformation through the acetylation of histones, also acetylates IN and controls its activity. We have found that p300 directly binds IN both in vitro and in the cells, as also specifically demonstrated by fluorescence resonance energy transfer technique analysis. This interaction results in the acetylation of three specific lysines (K264, K266, K273) in the carboxy-terminus of IN, a region that is required for DNA binding. Acetylation increases IN affinity to DNA, and promotes the DNA strand transfer activity of the protein. In the context of the viral replication cycle, point mutations in the IN acetylation sites abolish virus replication by specifically impairing its integration capacity. This is the first demonstration that HIV-1 IN activity is specifically regulated by post-translational modification.
| S-EPMC1201351 | biostudies-literature
Project description:The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3' half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly.
| S-EPMC4234376 | biostudies-literature
Project description:The internal N(6)-methyladenosine (m(6)A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m(6)A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m(6)A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m(6)A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4(+) T-cells. We further mapped the YTHDF1-3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1-3 in cells had the opposite effects. Moreover, silencing the m(6)A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m(6)A erasers increased Gag expression. Our findings suggest an important role of m(6)A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis.
| S-EPMC4961459 | biostudies-literature
Project description: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.
2020-06-11 | GSE142490 | GEO
Project description:We show that NAT10-ac4C axis significantly regulates cell fates. To identify the molecular mechanisms of NAT10-ac4C-ANP32B axis in cell-fate transitions, we construct shNAT10 and shANP32B hESCs. acRIP-seq of shCTR and shNAT10 hESCs. We profiled ATAC-seq in shCTR, shNAT10, and shANP32B hESCs. We profiled H3K4me3 and H3K27me3 modifications and the binding of ANP32B by CUT&Tag in shCTR and shNAT10 hESCs.
2023-11-11 | GSE210504 | GEO