The transcriptome-wide mapping of 2-methylthio-N6-isopentenyladenosine at single-base resolution by ReACT-seq
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ABSTRACT:
ORGANISM(S): Escherichia coli K-12
PROVIDER: GSE221930 | GEO | 2023/03/15
REPOSITORIES: GEO
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Project description:The transcriptome-wide mapping of 2-methylthio-N6-isopentenyladenosine at single-base resolution by ReACT-seq
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Project description:5-Formylcytidine (f5C) is one type of post-transcriptional RNA modifi-cations, which is known at the wobble position of tRNA in mitochon-dria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil (DHU), thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. Thousands of f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA. Reference build: S288C_reference_genome_R64-2-1_20150113
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Project description:D is an abundant RNA modification found in all kingdoms of life; yet our understanding of this modified base in mammals is lacking due to the lack of detecting methods. Until now, even for the most abundanct RNA speices with D, tRNA, we still do not have the profile of its sites. In this work, we introduce Chemical Reduction Assisted Cytosine Incorporation sequencing (CRACI), a powerful method that enables whole-transcriptome quantitative mapping of D modifications at single-base resolution.
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Project description:We report RBS-Seq, a new RNA bisulfite sequencing method enabling the sensitive and simultaneous detection of m5C, pseudouridine, and m1A at single-base resolution transcriptome-wide. For each, we detect ‘signature’ base mismatches (for m5C and m1A), or 1-2 base deletions (for pseudouridine) structurally explained by ribose ring-opened pseudouridine-mono-bisulfite adducts. Our profiles for pseudouridine reveal clear signatures at known sites in tRNAs and rRNAs, and provide hundreds of new targets in non-coding RNAs and mRNAs. However, our results diverge greatly from prior work, suggesting ~10-fold fewer m5C sites, and the absence of substantial m1A in mRNAs.
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Project description:5-Hydroxymethylcytosine (5hmC) is a crucial epigenetic mark that plays a key role in regulating various physiological and pathological processes. Although several methods have been developed to detect 5hmC, direct genome-wide mapping at single-base resolution remains highly desirable. Herein, we developed a double-stranded deamination sequencing (DDD-seq) method for genome-wide mapping of 5hmC at single-base resolution. This method utilizes the double-stranded DNA deaminase SsdAcat from Pseudomonas syringae, which efficiently deaminates cytosine (C), 5-methylcytosine (5mC), 5hmC, 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) in duplex DNA, but not glycosylated 5hmC (5gmC). In DDD-seq, C, 5mC, 5fC, and 5caC in dsDNA are deaminated by SsdAcat and read as T during sequencing, while 5gmC is resistant to deamination, allowing its identification by detecting the remaining C in sequence reads. The map of 5hmC generated by DDD-seq in mouse cerebellum tissue closely aligns with results obtained from the ACE-seq method. Additionally, applying DDD-seq to cerebellum tissue of mouse subjected to chronic sleep deprivation revealed significant global changes in 5hmC distribution on genomic DNA. In contrast to previous single-stranded deaminase APOBEC3A-based mapping methods that require denaturation of dsDNA into ssDNA, DDD-seq eliminates this step, reducing risks associated with incomplete denaturation and simplifying sequencing library construction. Additionally, SsdAcat demonstrates superior thermostability and activity across a wide range of pH values and temperatures, making DDD-seq applicable in broader scenarios with more accessible conditions. Collectively, the DDD-seq method is straightforward, bisulfite-free, and eliminates the need for DNA denaturation step, making it a valuable tool for direct and quantitative detection of 5hmC in genomes at single-base resolution.
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Project description:2’-O-methylation (Nm) is a prevalent post-transcriptional RNA modification present in many cellular RNAs and plays a critical role in modulating both the physical properties and regulation of eukaryotic RNAs. Studies of Nm modifications in RNA have long been hampered by a lack of effective mapping methods. Previously reported approaches can work well for detecting Nm modifications on abundant RNAs, but face challenges when applied to low-abundant RNAs, such as mRNA, lack stoichiometric information, and are challenged by issues of RNA sample degradation due to chemical treatment. Here, we present Nm-Mut-seq, a mutation signature-based Nm mapping method, which uses a custom reverse transcriptase (RT) that installs mutations at Am, Cm, and Gm-modified sites (Um is undetectable by this method). Our work provides a much-needed approach to detect Nm at base resolution in low abundant RNAs and to estimate the stoichiometry of each modified site transcriptome-wide.
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Project description:Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2’-O-Methylation of the ribose moiety is one of the most abundant post-transcriptional modifications of the RNA. We describe here a high-throughput method that enables fast and accurate mapping at single-base resolution, and quantitation, of 2’-OMe modified residues. Our approach expands the actual repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications.
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Project description:Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.
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Project description:Purpose:Detect transcriptome-wide mapping of m6A modifications by MeRIP-seq between Rhein and DMSO treated 3T3-L1 differentiation adipocytes at the preadipocytes, MCE stage and differentiation stage. Methods:Total RNA was extracted using TRIzol Reagent followed by chemical fragmentation. The fragmented RNA was incubated with anti-m6A antibody. The desired m6A-enriched RNA eluate was used to construct cDNA library in parallel with input control. Conclusion: Our study revealed the difference of m6A-methylated RNA between DMSO and Rhein treated cells by RNA-seq and MeRIP-seq technologies.
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