Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas.

Previous studies have suggested an important role for N6-methyladenosine (m6A) modification in the proliferation of glioma cells. N6, 2'-O-dimethyladenosine (m6Am) is another methylated form affecting the fate and function of most RNA. PCIF1 has recently been identified as the sole m6Am methyltransferase in mammalian mRNA. However, it remains unknown about the role of PCIF1 in the growth and survival of glioma cells. We constructed glioma cell lines that stably downregulated/upregulated PCIF1, established intracranial xenograft models using these cell lines, and employed the following methods for investigations: CCK-8, EdU, colony formation, flow cytometry, qRT-PCR, Western blot, and immunohistochemistry. Downregulating PCIF1 promoted glioma cell proliferation, while overexpressing PCIF1 showed the opposite effects. Overexpression of PCIF1 blocked cell cycle progression and induced apoptosis in glioma cells, which was further confirmed by alterations in the expression of cell checkpoint proteins and apoptotic markers. Interestingly, disruption of PCIF1 methyltransferase activity slightly reversed the effect of PCIF1 overexpression on cell proliferation, but had no significant reversal effects on cell cycle progression or apoptosis. Knockdown of PCIF1 promoted the growth of gliomas, while overexpressing PCIF1 inhibited tumor growth and prolonged the survival time of tumor-bearing mice. In addition, the mRNA and protein levels of PCIF1 were gradually decreased with the increase of WHO grade in glioma tissues, but there was no significant correlation with patient survival. These results indicated that PCIF1 played a suppressing role in glioma growth and survival, which may not entirely depend on its methyltransferase activity.

Gao S ，Zhou J ，Hu Z ，Zhang S ，Wu Y ，Musunuru PP ，Zhang T ，Yang L ，Luo X ，Bai J ，Meng Q ，Yu R ... -  《-》

m6Am methyltransferase PCIF1 negatively regulates ciliation by inhibiting BICD2 expression.

Xie S ，Kuang W ，Guo M ，Yang F ，Jin H ，Chen X ，Yi L ，Huo C ，Xu Z ，Lin A ，Liu W ，Mao J ，Shu Q ，Zhou T ... -  《-》

The cap-specific m6A methyltransferase, PCIF1/CAPAM, is dynamically recruited to the gene promoter in a transcription-dependent manner.

N 6-methyladenosine (m6A), the most abundant modification in eukaryotic mRNAs, plays an important role in mRNA metabolism and functions. When adenosine is transcribed as the first cap-adjacent nucleotide, it is methylated at the ribose 2'-O and N6 positions, thus generating N6, 2'-O-dimethyladenosine (m6Am). Phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1) is a novel cap-specific adenine N6-methyltransferase responsible for m6Am formation. As PCIF1 specifically interacts with the Ser5-phosphorylated CTD of RNA polymerase II (Pol II), which is a marker for the early phase of transcription, PCIF1 is speculated to be recruited to the early elongating Pol II. In this study, subcellular fractionation and immunofluorescence microscopy demonstrated that PCIF1 is mainly localized to the transcriptionally active chromatin regions in HeLa cells. Chromatin immunoprecipitation (ChIP) revealed that PCIF1 was predominantly localized to the promoter of a broad range of Pol II-transcribed genes, including several protein-coding genes and non-coding RNA genes. Moreover, PCIF1 accumulation on these promoters depended entirely on transcriptional activity and Ser5 phosphorylation of the CTD. These results suggest that PCIF1 dynamically localizes to the Pol II early in transcription and may efficiently catalyze N6-methylation of the first adenosine residue of nascent mRNAs cotranscriptionally.

Sugita A ，Kuruma S ，Yanagisawa N ，Ishiguro H ，Kano R ，Ohkuma Y ，Hirose Y ... -  《-》

PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression.

mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.

Sendinc E ，Valle-Garcia D ，Dhall A ，Chen H ，Henriques T ，Navarrete-Perea J ，Sheng W ，Gygi SP ，Adelman K ，Shi Y ... -  《-》

PCIF1, the only methyltransferase of N6,2-O-dimethyladenosine.

Wu Y ，Pu X ，Wu S ，Zhang Y ，Fu S ，Tang H ，Wang X ，Xu M ... -  《Cancer Cell International》