Interactions, localization, and phosphorylation of the m(6)A generating METTL3-METTL14-WTAP complex.

N6-methyladenine (m6A) is found on many eukaryotic RNAs including mRNAs. m6A modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates m6A on mRNAs. METTL3/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. Although recent crystal structures revealed how the catalytic MT-A70 domains of METTL3 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. Here, we used recombinant proteins and mapped binding surfaces within the METTL3/14-WTAP complex. Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Using an in vitro methylation assay, we confirm that monomeric METTL3 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METTL3/14 activity by contributing to RNA substrate binding. Our biochemical work identifies characteristic features of METTL3/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex.

Schöller E ，Weichmann F ，Treiber T ，Ringle S ，Treiber N ，Flatley A ，Feederle R ，Bruckmann A ，Meister G ... -  《-》

METTL3 regulates m6A in endometrioid epithelial ovarian cancer independently of METTl14 and WTAP.

N6-methyladenosine (m6A) RNA methylation, one of the common RNA modifications, has been determined to execute crucial functions in tumorigenesis and cancer development. The m6A "writers" including methyltransferase like 3 (METTL3), METTL14, and Wilms tumor 1-associated protein (WTAP) contribute to the m6A modification process initiation. However, the coordination of m6A methyltransferase complex is not fully understood in endometrioid epithelial ovarian cancer (EEOC). In this study, mRNA and protein levels of METTL3, METTL14, and WTAP were detected in 33 EEOC cases using quantitative polymerase chain reaction (qPCR), immunohistochemistry, and western blot analysis. The overall m6A methylation was detected by dot plot. The METTL3 expression and overall m6A level were elevated in EEOC tissues, while the expressions of METTL14 and WTAP have no significant difference in EEOC compared to the adjacent tissues. The expression of METTL3 was an independent factor that correlated with poor malignancy and survival of EEOC patients. Moreover, METTL3 knockdown in TOV-112D and CRL-11731D cells weakened the capability of cell proliferation and migration, and promoted cell apoptosis compared to negative control and cells with WTAP or METTL14 knockdown using CCK-8 assay, transwell assay, wound healing assay, and TUNEL assay. Furthermore, METTL3 knockdown also reduced m6A enrichment of the genes associated with ovarian cancer including EIF3C, AXL, CSF-1, FZD10 in TOV-112D, and CRL-11731D cells by RIP-qPCR assay. Taken together, the high expressed METTL3 indicated poor malignancy and survival of EEOC via modulating the aberrant m6A RNA methylation. METTL3-mediated m6A modification, independent of WTAP and METTL14, was considered as a novel mechanism underlying m6A modulation and a potential therapeutic target of EEOC.

Ma Z ，Li Q ，Liu P ，Dong W ，Zuo Y ... -  《-》

Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.

Ping XL ，Sun BF ，Wang L ，Xiao W ，Yang X ，Wang WJ ，Adhikari S ，Shi Y ，Lv Y ，Chen YS ，Zhao X ，Li A ，Yang Y ，Dahal U ，Lou XM ，Liu X ，Huang J ，Yuan WP ，Zhu XF ，Cheng T ，Zhao YL ，Wang X ，Rendtlew Danielsen JM ，Liu F ，Yang YG ... -  《-》

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation.

Han D ，Longhini AP ，Zhang X ，Hoang V ，Wilson MZ ，Kosik KS ... -  《-》

SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function.

Du Y ，Hou G ，Zhang H ，Dou J ，He J ，Guo Y ，Li L ，Chen R ，Wang Y ，Deng R ，Huang J ，Jiang B ，Xu M ，Cheng J ，Chen GQ ，Zhao X ，Yu J ... -  《-》