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Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.Nature Biotechnology Oct 2018Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize...
Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.
Topics: Adenine; CRISPR-Cas Systems; Codon; Cytidine; DNA; Gene Editing; HEK293 Cells; Humans
PubMed: 29813047
DOI: 10.1038/nbt.4172 -
Cell Dec 2018Generation of the "epitranscriptome" through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function....
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.
Topics: Acetylation; Cytidine; HeLa Cells; Humans; N-Terminal Acetyltransferase E; N-Terminal Acetyltransferases; Protein Biosynthesis; RNA, Messenger
PubMed: 30449621
DOI: 10.1016/j.cell.2018.10.030 -
The New England Journal of Medicine Feb 2022New treatments are needed to reduce the risk of progression of coronavirus disease 2019 (Covid-19). Molnupiravir is an oral, small-molecule antiviral prodrug that is... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
New treatments are needed to reduce the risk of progression of coronavirus disease 2019 (Covid-19). Molnupiravir is an oral, small-molecule antiviral prodrug that is active against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
METHODS
We conducted a phase 3, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of treatment with molnupiravir started within 5 days after the onset of signs or symptoms in nonhospitalized, unvaccinated adults with mild-to-moderate, laboratory-confirmed Covid-19 and at least one risk factor for severe Covid-19 illness. Participants in the trial were randomly assigned to receive 800 mg of molnupiravir or placebo twice daily for 5 days. The primary efficacy end point was the incidence hospitalization or death at day 29; the incidence of adverse events was the primary safety end point. A planned interim analysis was performed when 50% of 1550 participants (target enrollment) had been followed through day 29.
RESULTS
A total of 1433 participants underwent randomization; 716 were assigned to receive molnupiravir and 717 to receive placebo. With the exception of an imbalance in sex, baseline characteristics were similar in the two groups. The superiority of molnupiravir was demonstrated at the interim analysis; the risk of hospitalization for any cause or death through day 29 was lower with molnupiravir (28 of 385 participants [7.3%]) than with placebo (53 of 377 [14.1%]) (difference, -6.8 percentage points; 95% confidence interval [CI], -11.3 to -2.4; P = 0.001). In the analysis of all participants who had undergone randomization, the percentage of participants who were hospitalized or died through day 29 was lower in the molnupiravir group than in the placebo group (6.8% [48 of 709] vs. 9.7% [68 of 699]; difference, -3.0 percentage points; 95% CI, -5.9 to -0.1). Results of subgroup analyses were largely consistent with these overall results; in some subgroups, such as patients with evidence of previous SARS-CoV-2 infection, those with low baseline viral load, and those with diabetes, the point estimate for the difference favored placebo. One death was reported in the molnupiravir group and 9 were reported in the placebo group through day 29. Adverse events were reported in 216 of 710 participants (30.4%) in the molnupiravir group and 231 of 701 (33.0%) in the placebo group.
CONCLUSIONS
Early treatment with molnupiravir reduced the risk of hospitalization or death in at-risk, unvaccinated adults with Covid-19. (Funded by Merck Sharp and Dohme; MOVe-OUT ClinicalTrials.gov number, NCT04575597.).
Topics: Administration, Oral; Adolescent; Adult; Aged; Aged, 80 and over; Antiviral Agents; COVID-19; Cytidine; Double-Blind Method; Female; Humans; Hydroxylamines; Male; Middle Aged; SARS-CoV-2; Treatment Outcome; Viral Load; Young Adult; COVID-19 Drug Treatment
PubMed: 34914868
DOI: 10.1056/NEJMoa2116044 -
Clinical and Translational Medicine May 2022Dysregulation of the epitranscriptome causes abnormal expression of oncogenes in the tumorigenic process. Previous studies have shown that NAT10 can regulate mRNA...
BACKGROUND
Dysregulation of the epitranscriptome causes abnormal expression of oncogenes in the tumorigenic process. Previous studies have shown that NAT10 can regulate mRNA translation efficiency through RNA acetylation. However, the role of NAT10-mediated acetylation modification in bladder cancer remains elusive.
METHODS
The clinical value of NAT10 was estimated according to NAT10 expression pattern based on TCGA data set and the tumor tissue array. Acetylated RNA immunoprecipitation sequencing was utilized to explore the role of NAT10 in mRNA ac4C modification. Translation efficiency and mRNA stability assay were applied to study the effect of NAT10-deletion on target genes. The nude mouse model and genetically engineered mice were conducted to further verify the characteristics of NAT10 in promoting BLCA progression and regulating downstream targets.
RESULTS
NAT10 was essential for the proliferation, migration, invasion, survival and the stem-cell-like properties of bladder cancer cell lines. NAT10 was responsible for mRNA ac4C modification in BLCA cells, including BCL9L, SOX4 and AKT1. Deficient NAT10 in both xenograft and transgenic mouse models of bladder cancer reduced the tumor burden. Furthermore, acetylated RNA immunoprecipitation sequencing data and RNA immunoprecipitation qPCR results revealed that NAT10 is responsible for a set of ac4C mRNA modifications in bladder cancer cells. Inhibition of NAT10 led to a loss of ac4C peaks in these transcripts and represses the mRNA's stability and protein expression. Mechanistically, the ac4C reduction modification in specific regions of mRNAs resulting from NAT10 downregulation impaired the translation efficiency of BCL9L, SOX4 and AKT1 as well as the stability of BCL9L, SOX4.
CONCLUSIONS
In summary, these findings provide new insights into the dynamic characteristics of mRNA's post-transcriptional modification via NAT10-dependent acetylation and predict a role for NAT10 as a therapeutic target in bladder cancer.
HIGHLIGHTS
NAT10 is highly expressed in BLCA patients and its abnormal level predicts bladder cancer progression and low overall survival rate. NAT10 is necessary and sufficient for BLCA tumourigenic properties. NAT10 is responsible for ac4C modification of target transcripts, including BCL9L, SOX4 and AKT1. NAT10 may serve as an effective and novel therapeutic target for BLCA.
Topics: Animals; Cytidine; Humans; Mice; N-Terminal Acetyltransferases; RNA; RNA, Messenger; SOXC Transcription Factors; Urinary Bladder Neoplasms
PubMed: 35522942
DOI: 10.1002/ctm2.738 -
Nature Structural & Molecular Biology Sep 2021Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the...
Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-D-N-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.
Topics: Animals; Antiviral Agents; Base Sequence; COVID-19; Cytidine; Humans; Hydroxylamines; Models, Molecular; Molecular Structure; Mutagenesis; Mutation; Nucleic Acid Conformation; Protein Binding; Protein Conformation; RNA, Viral; RNA-Dependent RNA Polymerase; SARS-CoV-2; Virus Replication; COVID-19 Drug Treatment
PubMed: 34381216
DOI: 10.1038/s41594-021-00651-0 -
Frontiers in Immunology 20225-Methylcytidine (m5C) methylation is an emerging epigenetic modification in recent years, which is associated with the development and progression of various cancers....
BACKGROUND
5-Methylcytidine (m5C) methylation is an emerging epigenetic modification in recent years, which is associated with the development and progression of various cancers. However, the prognostic value of m5C regulatory genes and the correlation between m5C methylation and the tumor microenvironment (TME) in prostate cancer remain unknown.
METHODS
In the current study, the genetic and transcriptional alterations and prognostic value of m5C regulatory genes were investigated in The Cancer Genome Atlas and Gene Expression Omnibus datasets. Then, an m5C prognostic model was established by LASSO Cox regression analysis. Gene set variation analyses (GSVA), gene set enrichment analysis (GSEA), clinical relevance, and TME analyses were conducted to explain the biological functions and quantify the TME scores between high-risk and low-risk subgroups. m5C regulatory gene clusters and m5C immune subtypes were identified using consensus unsupervised clustering analysis. The Cell-type Identification By Estimating Relative Subsets of RNA Transcripts algorithm was used to calculate the contents of immune cells.
RESULTS
was upregulated at transcriptional levels in PCa compared with normal tissues, and a high expression was associated with poor prognosis. An m5C prognostic model consisting of 3 genes (, , and ) was developed and a nomogram was constructed for improving the clinical applicability of the model. Functional analysis revealed the enrichment of pathways and the biological processes associated with RNA regulation and immune function. Significant differences were also found in the expression levels of m5C regulatory genes, TME scores, and immune cell infiltration levels between different risk subgroups. We identified two distinct m5C gene clusters and found their correlation with patient prognosis and immune cell infiltration characteristics. Naive B cells, CD8+ T cells, M1 macrophages and M2 macrophages were obtained and 2 m5C immune subtypes were identified. , , , and were differentially expressed between immune subtypes. The expression of was found to be correlated with the degree of immune cell infiltration.
CONCLUSIONS
Our comprehensive analysis of m5C regulatory genes in PCa demonstrated their potential roles in the prognosis, clinical features, and TME. These findings may improve our understanding of m5C regulatory genes in the tumor biology of PCa.
Topics: Cytidine; Genes, Regulator; Humans; Male; Methylation; Prostatic Neoplasms; RNA; Tumor Microenvironment
PubMed: 35757739
DOI: 10.3389/fimmu.2022.914577 -
Nucleic Acids Research Nov 2011In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs....
In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.
Topics: Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Cytidine; Genetic Therapy; HEK293 Cells; Humans; Mice; Protein Biosynthesis; Pseudouridine; RNA, Double-Stranded; RNA, Messenger; Transcription, Genetic; Transfection
PubMed: 21890902
DOI: 10.1093/nar/gkr695 -
Nucleic Acids Research Jan 2021The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains...
The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.
Topics: 3' Untranslated Regions; Animals; Base Sequence; Cell Line, Tumor; Codon Usage; Consensus Sequence; Cytidine; Embryonic Stem Cells; Gene Knockout Techniques; Genes, Reporter; HEK293 Cells; Humans; Immunoprecipitation; Methylation; Mice; Mice, Knockout; Mutagenesis, Site-Directed; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcriptome; tRNA Methyltransferases
PubMed: 33330931
DOI: 10.1093/nar/gkaa1193 -
Clinical and Translational Medicine Sep 2022N-4 cytidine acetylation (ac4C) is an epitranscriptomics modification catalyzed by N-acetyltransferase 10 (NAT10); important for cellular mRNA stability, rRNA...
BACKGROUND
N-4 cytidine acetylation (ac4C) is an epitranscriptomics modification catalyzed by N-acetyltransferase 10 (NAT10); important for cellular mRNA stability, rRNA biogenesis, cell proliferation and epithelial to mesenchymal transition (EMT). However, whether other crucial pathways are regulated by NAT10-dependent ac4C modification in cancer cells remains unclear. Therefore, in this study, we explored the impact of NAT10 depletion in cancer cells using unbiased RNA-seq.
METHODS
High-throughput sequencing of knockdown NAT10 in cancer cells was conducted to identify enriched pathways. Acetylated RNA immunoprecipitation-seq (acRIP-seq) and RIP-PCR were used to map and determine ac4C levels of RNA. Exogenous palmitate uptake assay was conducted to assess NAT10 knockdown cancer cells using Oil Red O staining and lipid content analysis. Gas-chromatography-tandem mass spectroscopy (GC/MS) was used to perform untargeted lipidomics.
RESULTS
High-throughput sequencing of NAT10 knockdown in cancer cells revealed fatty acid (FA) metabolism as the top enriched pathway through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in differentially downregulated genes. FA metabolic genes such as ELOLV6, ACSL1, ACSL3, ACSL4, ACADSB and ACAT1 were shown to be stabilised via NAT10-dependent ac4C RNA acetylation. Additionally, NAT10 depletion was shown to significantly reduce the levels of overall lipid content, triglycerides and total cholesterol. Further, NAT10 depletion in palmitate-loaded cancer cells showed decrease in ac4C levels across the RNA transcripts of FA metabolic genes. In untargeted lipidomics, 496 out of 2 279 lipids were statistically significant in NAT10 depleted cancer cells, of which pathways associated with FA metabolism are the most enriched.
CONCLUSIONS
Conclusively, our results provide novel insights into the impact of NAT10-mediated ac4C modification as a crucial regulatory factor during FA metabolism and showed the benefit of targeting NAT10 for cancer treatment.
Topics: Acetyltransferases; Cholesterol; Cytidine; Epithelial-Mesenchymal Transition; Fatty Acids; Neoplasms; Palmitates; RNA; Transferases; Triglycerides
PubMed: 36149760
DOI: 10.1002/ctm2.1045 -
Nucleic Acids Research Oct 2022Post-transcriptional RNA modifications critically regulate various biological processes. N4-acetylcytidine (ac4C) is an epi-transcriptome, which is highly conserved in...
Post-transcriptional RNA modifications critically regulate various biological processes. N4-acetylcytidine (ac4C) is an epi-transcriptome, which is highly conserved in all species. However, the in vivo physiological functions and regulatory mechanisms of ac4C remain poorly understood, particularly in mammals. In this study, we demonstrate that the only known ac4C writer, N-acetyltransferase 10 (NAT10), plays an essential role in male reproduction. We identified the occurrence of ac4C in the mRNAs of mouse tissues and showed that ac4C undergoes dynamic changes during spermatogenesis. Germ cell-specific ablation of Nat10 severely inhibits meiotic entry and leads to defects in homologous chromosome synapsis, meiotic recombination and repair of DNA double-strand breaks during meiosis. Transcriptomic profiling revealed dysregulation of functional genes in meiotic prophase I after Nat10 deletion. These findings highlight the crucial physiological functions of ac4C modifications in male spermatogenesis and expand our understanding of its role in the regulation of specific physiological processes in vivo.
Topics: Male; Mice; Animals; Meiosis; Cytidine; Chromosome Pairing; Germ Cells; Mammals
PubMed: 35801907
DOI: 10.1093/nar/gkac594