Gene expression in Mon-Mac-6 cells treated with METTL3 PROTAC, WD6305
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE234985 | GEO | 2023/11/29
REPOSITORIES: GEO
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Project description:N6-methyladenosine (m6A) methylation is the most prevalent RNA modification, which plays a critical role in various bioprocesses.1 This modification is predominantly catalyzed by the m6A methyltransferase complex (MTC), consisting of core subunits such as methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14), along with cofactors like Wilms tumor 1-associated protein (WTAP). We described the development of a first-in-class potent and selective METTL3-targeted degrader, WD6305, which can effectively degrade both METTL3 and its partner protein METTL14. WD6305 showed superior antiproliferative effects in multiple AML cell lines compared to its parent inhibitor, UZH2. Here, we want to compare the changes in genes between the two.
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Project description:label the cells overexpressed Myc tagged METTL3 and Flag tagged WTAP with 4-SU, the RNA bound by METT3,WTAP can be got by Myc or Flag IP followed by RNA isolation by using the TRIzol (Invitrogen) reagent by following the company manual.the RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 50 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane. discovery of the binding motif of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells
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Project description:Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins which undermines the growth and survival of AML cells. However, BETi treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4, which potentially compromises the activity of BETi in AML cells. Unlike BETi, BET-PROTAC (proteolysis-targeting chimera) ARV-825 recruits and utilize an E3-ubiquitin ligase to effectively degrade BETPs in AML cells. BET-PROTACs induce more apoptosis than BETi of mtRUNX1 AML cells. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi. It was noted that treatment with BETi or BET-PROTAC caused significant and sustained depletion of RUNX1 in AML cells. We also determined the effects of global depletion of RUNX1 in mtRUNX1 expressing AML OCI-AML5 cells. We observed an overlap in the signature of RUNX1 knockdown by shRNA with that of OTX015 and ARV-825 in OCI-AML5 cells.
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Project description:N6-methyladenosine (m6A) methylation of mRNA by the methyltransferase complex (MTC), with core components including METTL3-METTL14 heterodimers and Wilms’ tumor 1-associated protein (WTAP), contributes to breast tumorigenesis, but the mechanism of MTC assembly remains elusive. Here, we identify a novel cleaved form METTL3a (residues 239-580 of METTL3), that is highly expressed in breast cancer. Furthermore, we find that both METTL3a and full-length METTL3 are required for MTC assembly, RNA m6A deposition, as well as cancer cell proliferation. Mechanistically, we find that METTL3a is required for METTL3-METTL3 interaction, which is a prerequisite step for recruitment of WTAP in MTC assembly. Analysis of m6A sequencing data shows that depletion of METTL3a globally disrupts m6A methylation, and METTL3a mediates mTOR activation via m6A-mediated suppression of TMEM127 expression. Consequently, we find that METTL3 cleavage is mediated by proteasome in an mTOR-dependent manner, revealing positive regulatory feedback between METTL3a and mTOR signaling. Our findings reveal METTL3a as an important component for MTC assembly, and suggest the METTL3a-mTOR axis as a potential therapeutic target for breast cancer.
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Project description:Scientists have developed several methods to control the activity of Cas protein in a timely way and hence reduce off-target effects, including anti-CRISPR proteins and small molecule inhibitors. The PROTAC is a new conception of using the natural ubiquitin-proteasome system to degrade the protein of interest in drug design and development. Here, we engineered Cas proteins (Cas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (also known as the π-clamp system) and demonstrate that the modified CasFCPF proteins could be labeled in live cells by perfluoroaromatics carrying FITC fluorophore or were degraded by perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). Proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed high target specificity, suggesting a wide application of chemically induced proximity in combination with site-specific protein modification in the regulation of protein stability, activity and functionality.
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Project description:METTL3 and METTL14 are considered to faithfully form the m6A writing complex in a 1:1 ratio, regulating the fate of mRNA by adding m6A modifications. However, recent studies have shown inconsistent expression and prognostic value of METTL3 and METTL14 in some tumors, suggesting that they may not be faithful in tumors. Pan-cancer analysis based on TCGA data reveals significant differences in expression, function, tumor burden correlation, and immune correlation between METTL3 and METTL14, especially in esophageal squamous cell carcinoma (ESCC). Knockdown of METTL3 significantly inhibits the cell proliferation in vitro and in vivo in ESCC EC109 cells, while the impact of METTL14 knockdown on proliferation is limited, and it cannot abolish the expression of METTL3 protein. mRNA-seq results indicate that METTL3 independently regulates the expression of 1615 genes, while only 776 genes are co-regulated by METTL3 and METTL14. Furthermore, through immunofluorescence co-localization, it is observed that METTL3 and METTL14 have certain inconsistencies in cellular localization. HPLC-MS results show that METTL3 independently binds to the Nop56p-associated pre-rRNA complex and mRNA splicing complex, separate from METTL14. Through bioinformatics and various omics studies, we have preliminarily discovered that METTL3 independently regulating tumor cell proliferation, and the participation in mRNA splicing may be a critical molecular mechanism. Our study provides an experimental basis and theoretical foundation for further understanding of the m6A writing complex and tumor therapy targeting METTL3.
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