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Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

ABSTRACT: Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

PROVIDER: PRJNA503400 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8145460.fastq.gz	Fastqsanger.gz
	SRR8145461.fastq.gz	Fastqsanger.gz
	SRR8145462.fastq.gz	Fastqsanger.gz
	SRR8145463.fastq.gz	Fastqsanger.gz
	SRR8145464.fastq.gz	Fastqsanger.gz

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Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

Project description:N6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2'-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.

2018-11-02 | GSE122071 | GEO

Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. PAR-CLIP and m6A-seq in HeLa cells

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mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription

Project description:mRNA cap addition occurs early during RNA pol II transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription, independently of mRNA capping and translation. In cells, sub-lethal suppression of RNMT-RAM reduces RNA pol II occupancy, net mRNA synthesis and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independently of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA-binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription associated factors including RNA pol II subunits, SPT4, SPT6 and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2B K120 ubiquitination, H3 K4 and K36 methylation, RNA pol II S5 and S2 phosphorylation and PAFc recruitment. These findings suggest that multiple interactions between RNMT-RAM, RNA pol II factors and RNA along the transcription unit stimulate transcription.

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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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CK2 phosphorylation of cap methyltransferase CMTR1 promotes RNAPII interaction

Project description:CK2 phosphorylation of cap methyltransferase CMTR1 promotes RNAPII interaction

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Project description:After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. Here we used chromatin immunoprecipitation (ChIP) to elucidate the general initiation-elongation transition of Pol II in yeast. We extended our ChIP-chip occupancy profiling to capping enzymes and the cap-binding complex (CBC). We found that the first two capping enzymes, Cet1 and Ceg1, are recruited near the TSS, whereas the third enzyme, the methyltransferase Abd1, is recruited about 110 nt downstream of the TSS, and CBC is recruited further downstream. ChIP was performed using TAP-tagged S. cerevisiae strains (see protocols).

2013-07-30 | E-MTAB-1552 | biostudies-arrayexpress

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

Project description:The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

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mRNA cap methylation in pluripotency and differentiation

Project description:The mRNA cap recruits factors essential for transcript processing and translation initiation. We report that regulated mRNA cap methylation is a feature of embryonic stem cell (ESC) differentiation. Expression of the mRNA cap methyltransferase activating subunit, RAM, is elevated in ESCs resulting in high levels of mRNA cap methylation and expression of Oct4 and Sox2 and other pluripotency-associated factors. During neural differentiation, RAM is suppressed which is required for loss of Oct4 and Sox2 and correct expression of neural markers. Cells were treated with control or RAM siRNA and cytosolic RNA or polysome RNA (actively translated) was sequenced.

2016-06-14 | E-GEOD-81849 | biostudies-arrayexpress

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

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

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