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M5C methyltransferase NSUN3 regulates variant gene expression and development in blood-stage Plasmodium falciparum [RIP-seq]

ABSTRACT: m5C methyltransferase NSUN3 regulates variant gene expression and development in blood-stage Plasmodium falciparum [RIP-seq]

PROVIDER: PRJNA1139780 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29967051_1.fastq.gz	Fastqsanger.gz
	SRR29967051_2.fastq.gz	Fastqsanger.gz

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m5C methyltransferase NSUN3 regulates variant gene expression and development in blood-stage Plasmodium falciparum [RIP-seq]

Project description:C5 methylation of cytosine is a relatively abundant post-transcriptional modification in eukaryotes, and the NSUN family has been identified as an important executor of m5C modification. Currently, little is known about this family in Plasmodium spp. Therefore, we constructed a pfnsun3 gene knockdown strain and the knockdown efficiency was confirmed through growth curves and western blot experiments. The target genes of PfNSUN3 was obtained by RNA immunoprecipitation and high-throughput transcriptome sequencing experiments. Our data reveal that pfnsun3 is an indispensable post-transcriptional RNA modification in regulating variant gene expression in Plasmodium falciparum.

2025-04-08 | GSE273004 | GEO

m5C methyltransferase NSUN3 regulates variant gene expression and development in blood-stage Plasmodium falciparum [RNA-seq]

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m5C methyltransferase NSUN3 regulates variant gene expression and development in blood-stage Plasmodium falciparum [RNA-seq]

Project description:m5C methyltransferase NSUN3 regulates variant gene expression and development in blood-stage Plasmodium falciparum [RNA-seq]

| PRJNA1139782 | ENA

RNA m5C Methylation in breast cancer using MeRIP-Seq

Project description:RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody. Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700). m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA. This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.

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Profiling of 5-methylcytosine (m5C) methylation in Arabidopsis thaliana wild-type

Project description:Methylation of carbon 5 in cytosine (5-methylcytosine; m5C) is a well-characterized DNA modification, and is also predominantly reported in highly abundant noncoding RNAs, such as rRNA and tRNA, in both prokaryotes and eukaryotes. However, the distribution and biological functions of m5C in plant mRNAs remain largely unknown. Here we develop an m5C RNA immunoprecipitation followed by deep sequencing approach (m5C-RIP-seq) to achieve transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identifies 6,045 putative m5C peaks in 4,465 expressed genes in young seedlings. m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further show that a RNA (cytosine-5)-methyltransferase, tRNA specific methyltransferase 4B (TRM4B), exhibits the m5C mRNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m5C levels in root mRNA. Furthermore, TRM4B affects transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels. Our results suggest that m5C in mRNA is a new epitranscriptome marker widely distributed in plant genes, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.

2017-10-05 | GSE94022 | GEO

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2024-06-05 | GSE249045 | GEO

Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs using dCasRx conjugated methyltransferase and demethylase

Project description:5-methylcytosine, an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification systems (termed ‘RCMS’), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localised dCasRx conjugated to either to a methyltransferase, NSUN2/NSUN6 or a demethylase, the catalytic domain of mouse Tet2 (Tet2 CD), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within tRNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

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NSUN2 promotes T-ALL cell growth as anRNA m5C methyltransferase

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