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CROWN-seq profiling of snRNA m6Am modification in pig ST cells upon PRV infection

ABSTRACT: CROWN-seq profiling of snRNA m6Am modification in pig ST cells upon PRV infection

PROVIDER: PRJNA1139787 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29967054_1.fastq.gz	Fastqsanger.gz
	SRR29967054_2.fastq.gz	Fastqsanger.gz
	SRR29967055_1.fastq.gz	Fastqsanger.gz
	SRR29967055_2.fastq.gz	Fastqsanger.gz
	SRR29967056_1.fastq.gz	Fastqsanger.gz

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CROWN-seq profiling of snRNA m6Am modification in pig ST cells upon PRV infection

Project description:Fat mass and obesity-associated protein (FTO) was discovered as the first RNA demethylase and mediates the demethylation of N6,2′-O-dimethyladenosine (m6Am), a prevalent RNA modification adjacent to the 5’ cap. Despite its significance, the regulation of the m6Am demethylase and the potential impact of virus infection on FTO remain largely unexplored. In this study, we show that the alphaherpesvirus pseudorabies virus (PRV) triggers phosphorylation of FTO and that this phosphorylation is caused by the viral serine/threonine protein kinase UL13. To test whether the phosphorylated FTO can result in different demethylation on snRNA, we performed CROWN-seq to quantify m6Am stoichiometry in cells.

2024-12-02 | GSE273011 | GEO

Absolute transcription-start nucleotide m6Am stoichiometry quantification by CROWN-Seq

Project description:The 5’ end RNA molecules transcribed by RNA polymerase II are known to be modified in human cells. Specifically, A-initiated transcripts can be methylated at both the 2’-O position in the ribose (2’-O-methylation, or cap-1) and the N6-position in the base (N6-methylation). Almost all A-initiated transcripts have the cap-1 modification, however, the N6-methylation is optional. As a result, A-initiated transcripts in the cell can be Am (cap-1 only) or m6Am (cap-1 and N6-methylation) modified. To study m6Am, we developed CROWN-seq, which simultaneously obtains the transcription-start information and the stoichiometry of N6-methyl. With CROWN-seq, we found that the previous m6Am mapping studies highly underestimated the diversity of the transcription-start site (nucleotide) of the transcriptome. In this study, we used CROWN-seq to reveal m6Am sites and stoichiometry among different human cell lines. We demonstrated that m6Am stoichiometry in mRNA is in general high, however, m6Am stoichiometry in snRNA, snoRNA, and their pseudogenes is more variable.

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METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

Project description:N6-methylation of 2’-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2’-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as overall 3’ splice-site weakness with certain motif positions more affected than others. This study establishes that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

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Absolute transcription-start nucleotide m6Am stoichiometry quantification by CROWN-Seq

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PrV/PK15 kinetics_SLA/PrV slides

Project description:To study in vitro the epithelial cells and PrV interactions during infection, we followed PrV and PK15 cells transcriptome modifications during time-course infection (I) and mock-infection (MI).Six time points were studied: just after I and MI, 1h, 2h, 4h, 8h and 12h post-I and MI. For this study, a pig DNA/cDNA microarray containing genes of the SLA region, additional genes encoding other important immunological molecules and all the PrV genes was constructed. Keywords: infection time course

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PrV/PK15 kinetics_QiagenNRSP8 slides

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PrV/PK15 kinetics_QiagenNRSP8 slides

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METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

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