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M6Am-Exo-Seq mapping of MEL624 control and PCIF1 KO cells

ABSTRACT: m6Am-Exo-Seq mapping of MEL624 control and PCIF1 KO cells

PROVIDER: PRJNA506410 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8234010.fastq.gz	Fastqsanger.gz
	SRR8234020.fastq.gz	Fastqsanger.gz
	SRR8234030.fastq.gz	Fastqsanger.gz
	SRR8234011.fastq.gz	Fastqsanger.gz
	SRR8234021.fastq.gz	Fastqsanger.gz

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m6Am-Exo-Seq mapping of MEL624 control and PCIF1 KO cells

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HIV reprograms host m6Am RNA methylome by viral Vpr protein-mediated degradation of PCIF1

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Project description:mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2′-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to “internal” sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am’s function.

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m6Am RNA methylation enzyme PCIF1 restricts HIV infection and is degraded by viral Vpr protein

Project description:To identify m6Am genes that are responsible for HIV inhibition, MeRIP-seq was performed in control and PCIF1 KO T cells infected with HIV.

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The mammalian cap-specific m6Am RNA methyltransferase PCIF1 regulates transcript levels in mouse tissues

Project description:The 5' end of eukaryotic mRNAs is protected by the m7G-cap structure. The transcription start site nucleotide is ribose methylated (Nm) in many eukaryotes, while an adenosine at this position is further methylated at the N6 position (m6A) by the mammalian Phosphorylated CTD-interacting Factor 1 (PCIF1) to generate m6Am. Here we show that while loss of cap-specific m6Am in mice does not affect viability or fertility, the Pcif1 mutants display reduced body-weight. Transcriptome analyses of mutant mouse tissues support a role for the cap-specific m6Am modification in stabilizing transcripts. In contrast, the Drosophila Pcif1 is catalytically-dead, but like its mammalian counterpart it retains the ability to associate with the Ser5-phosphorylated CTD of RNA pol II. Finally, we show that the Trypanosoma Pcif1 is an m6Am methylase that contributes to the N6,N6,2?-O-trimethyladenosine (m62Am) in the hypermethylated cap4 structure of Trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.

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Project description:Purpose: To identify the in vivo RNA targets of PCIF1, we performed meRIP experiments for PCIF1 KD and control cells using an anti-m6A antibody. We envisioned that the cap-specific PCIF1 should selectively alter the m6Am modification at the 5-terminal region of transcripts. Indeed, we observed a reduction of modification peaks at the 5’-end but not the 3’-UTR regions of mRNA upon PCIF1 KD.

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Project description:We analyzed m6A distribution by m6A MeRIP in otder to determine the effect of PCIF1 KO over m6A distribution in human melanoma cells

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Project description:PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression

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Project description:We previously identified PCIF1 (Phosphorylated CTD Interacting Factor 1) as a novel phosphorylated C-terminal domain (CTD) of RNA polymerase II. We also recently identified PCIF1 as a new cap-specific adenine N6 methyltransferase (CAPAM) responsible for creating N6, 2’-O-dimethyladenosine (m6Am) at the 5’-end of mRNAs. However, it remains unclear how PCIF1 regulates gene expression. To identify genes whose expression levels are affected by PCIF1 knockdown in human cultured cells, we performed gene expression profiling by microarray analysis.

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