METTL14-mediated N6-methyladenosine modification of SOX4 mRNA inhibits tumor metastasis in colorectal cancer.
ABSTRACT: BACKGROUND:Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC. METHODS:Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action. RESULTS:METTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals. CONCLUSIONS:Decreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.
Project description:The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.
Project description:The importance of RNA methylation in biological processes is an emerging focus of investigation. We report that altering m6A levels by silencing either N 6-adenosine methyltransferase METTL14 (methyltransferase-like 14) or demethylase ALKBH5 (ALKB homolog 5) inhibits cancer growth and invasion. METTL14/ALKBH5 mediate their protumorigenic function by regulating m6A levels of key epithelial-mesenchymal transition and angiogenesis-associated transcripts, including transforming growth factor-? signaling pathway genes. Using MeRIP-seq (methylated RNA immunoprecipitation sequencing) analysis and functional studies, we find that these target genes are particularly sensitive to changes in m6A modifications, as altered m6A status leads to aberrant expression of these genes, resulting in inappropriate cell cycle progression and evasion of apoptosis. Our results reveal that METTL14 and ALKBH5 determine the m6A status of target genes by controlling each other's expression and by inhibiting m6A reader YTHDF3 (YTH N 6-methyladenosine RNA binding protein 3), which blocks RNA demethylase activity. Furthermore, we show that ALKBH5/METTL14 constitute a positive feedback loop with RNA stability factor HuR to regulate the stability of target transcripts. We discover that hypoxia alters the level/activity of writers, erasers, and readers, leading to decreased m6A and consequently increased expression of target transcripts in cancer cells. This study unveils a previously undefined role for m6A in cancer and shows that the collaboration among writers-erasers-readers sets up the m6A threshold to ensure the stability of progrowth/proliferation-specific genes, and protumorigenic stimulus, such as hypoxia, perturbs that m6A threshold, leading to uncontrolled expression/activity of those genes, resulting in tumor growth, angiogenesis, and progression.
Project description:Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer?related death among women worldwide. Evidence indicates that posttranscriptional N6?methyladenosine (m6A) modification modulates BC development. In the present study, we assessed BC and normal tissues to investigate this connection. RNA m6A levels were determined by methylation quantification assay. The effects of methyltransferase?like 14 (METTL14) gain?of?expression or co?transfection with an m6A inhibitor on cell migration and invasion abilities were determined by Transwell assays. The levels of differentially expressed (DE) miRNAs were verified by real?time quantitative PCR (RT?qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) were performed to analyze potential function of target genes of the DE miRNAs. The effects of candidate miRNAs modulated by METTL14 on cell migration and invasion abilities were confirmed by Transwell assays. We demonstrated that m6A methyltransferase METTL14 was significantly upregulated in BC tissues compared with normal tissues. METTL14 gain? and loss?of?expression regulated m6A levels in MCF?7 and MDA?MB?231 cells. The cell function assays revealed that METTL14 overexpression enhanced the migration and invasion capacities of BC cells. Moreover, treatment with the m6A inhibitor suppressed this enhanced cell migration and invasion. Additionally, aberrant expression of METTL14 reshaped the miRNA profile in BC cell lines. The remodeled DE miRNA/mRNA network was found to be most enriched in cancer pathways, and DE miRNAs were enriched in cell adhesion terms. hsa?miR?146a?5p modulated by METTL14 promoted cell migration and invasion. METTL14 modulates m6A modification and hsa?miR?146a?5p expression, thereby affecting the migration and invasion of breast cancer cells.
Project description:BACKGROUND:As one of the most frequent chemical modifications in eukaryotic mRNAs, N6-methyladenosine (m6A) modification exerts important effects on mRNA stability, splicing, and translation. Recently, the regulatory role of m6A in tumorigenesis has been increasingly recognized. However, dysregulation of m6A and its functions in tumor epithelial-mesenchymal transition (EMT) and metastasis remain obscure. METHODS:qRT-PCR and immunohistochemistry were used to evaluate the expression of methyltransferase-like 3 (METTL3) in gastric cancer (GC). The effects of METTL3 on GC metastasis were investigated through in vitro and in vivo assays. The mechanism of METTL3 action was explored through transcriptome-sequencing, m6A-sequencing, m6A methylated RNA immunoprecipitation quantitative reverse transcription polymerase chain reaction (MeRIP qRT-PCR), confocal immunofluorescent assay, luciferase reporter assay, co-immunoprecipitation, RNA immunoprecipitation and chromatin immunoprecipitation assay. RESULTS:Here, we show that METTL3, a major RNA N6-adenosine methyltransferase, was upregulated in GC. Clinically, elevated METTL3 level was predictive of poor prognosis. Functionally, we found that METTL3 was required for the EMT process in vitro and for metastasis in vivo. Mechanistically, we unveiled the METTL3-mediated m6A modification profile in GC cells for the first time and identified zinc finger MYM-type containing 1 (ZMYM1) as a bona fide m6A target of METTL3. The m6A modification of ZMYM1 mRNA by METTL3 enhanced its stability relying on the "reader" protein HuR (also known as ELAVL1) dependent pathway. In addition, ZMYM1 bound to and mediated the repression of E-cadherin promoter by recruiting the CtBP/LSD1/CoREST complex, thus facilitating the EMT program and metastasis. CONCLUSIONS:Collectively, our findings indicate the critical role of m6A modification in GC and uncover METTL3/ZMYM1/E-cadherin signaling as a potential therapeutic target in anti-metastatic strategy against GC.
2019-01-01 | S-EPMC6790244 | BioStudies
Project description:The N6-methyladenosine (m6A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6A methyltransferase complex, as a novel pathway that controls m6A-deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C-terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit significantly reduced global m6A levels. Transcriptome-wide m6A analysis identified 1,701 METTL14 arginine methylation-dependent m6A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation-dependent m6A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6A regulation and new functions of arginine methylation in RNA metabolism.
Project description:N6-methyladenosine (m6A) is an abundant modification in messenger RNA and noncoding RNAs that affects RNA metabolism. Methyltransferase-like protein 16 (METTL16) is a recently confirmed m6A RNA methyltransferase that methylates U6 spliceosomal RNA and interacts with the 3'-terminal RNA triple helix of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1). Here, we present two X-ray crystal structures of the N-terminal methyltransferase domain (residues 1-291) of human METTL16 (METTL16_291): an apo structure at 1.9 Å resolution and a post-catalytic S-adenosylhomocysteine-bound complex at 2.1 Å resolution. The structures revealed a highly conserved Rossmann fold that is characteristic of Class I S-adenosylmethionine-dependent methyltransferases and a large, positively charged groove. This groove likely represents the RNA-binding site and it includes structural elements unique to METTL16. In-depth analysis of the active site led to a model of the methyl transfer reaction catalyzed by METTL16. In contrast to the major m6A methyltransferase heterodimer METTL3/METTL14, full-length METTL16 forms a homodimer and METTL16_291 exists as a monomer based on size-exclusion chromatography. A native gel-shift assay shows that METTL16 binds to the MALAT1 RNA triple helix, but monomeric METTL16_291 does not. Our results provide insights into the molecular structure of METTL16, which is distinct from METTL3/METTL14.
Project description:BACKGROUND:Colorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and methyltransferase-like 3 (METTL3) participates in tumor progression in several cancer types. However, its role in CRC remains unexplored. METHODS:Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect METTL3 expression in cell lines and patient tissues. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3. The biological functions of METTL3 were investigated in vitro and in vivo. RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. RNA stability assay was used to detect the half-lives of the downstream genes of METTL3. RESULTS:Using TCGA database, higher METTL3 expression was found in CRC metastatic tissues and was associated with a poor prognosis. MeRIP-seq revealed that SRY (sex determining region Y)-box 2 (SOX2) was the downstream gene of METTL3. METTL3 knockdown in CRC cells drastically inhibited cell self-renewal, stem cell frequency and migration in vitro and suppressed CRC tumorigenesis and metastasis in both cell-based models and PDX models. Mechanistically, methylated SOX2 transcripts, specifically the coding sequence (CDS) regions, were subsequently recognized by the specific m6A "reader", insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), to prevent SOX2 mRNA degradation. Further, SOX2 expression positively correlated with METTL3 and IGF2BP2 in CRC tissues. The combined IHC panel, including "writer", "reader", and "target", exhibited a better prognostic value for CRC patients than any of these components individually. CONCLUSIONS:Overall, our study revealed that METTL3, acting as an oncogene, maintained SOX2 expression through an m6A-IGF2BP2-dependent mechanism in CRC cells, and indicated a potential biomarker panel for prognostic prediction in CRC.
Project description:BACKGROUND:Pancreatic cancer is one of the most lethal human cancers. N6-methyladenosine (m6A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive. METHODS:LC/MS was used to profile m6A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m6A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14. RESULTS:We found that the m6A levels were elevated in approximately 70% of the pancreatic cancer samples. Furthermore, we demonstrated that METTL14 is the major enzyme that modulates m6A methylation (frequency and site of methylation). METTL14 overexpression markedly promoted pancreatic cancer cell proliferation and migration both in vitro and in vivo, via direct targeting of the downstream PERP mRNA (p53 effector related to PMP-22) in an m6A-dependent manner. Methylation of the target adenosine lead to increased PERP mRNA turnover, thus decreasing PERP (mRNA and protein) levels in pancreatic cancer cells. CONCLUSIONS:Our data suggest that the upregulation of METTL14 leads to the decrease of PERP levels via m6A modification, promoting the growth and metastasis of pancreatic cancer; therefore METTL14 is a potential therapeutic target for its treatment.
Project description:N6-methyladenosine (m6A) RNA methylation is the most prevalent modification of messenger RNAs (mRNAs) and catalyzed by a multicomponent methyltransferase complex (MTC), among which methyltransferase-like 3 (METTL3) and METTL14 are two core molecules. However, METTL3 and METTL14 play opposite regulatory roles in hepatocellular carcinoma (HCC). Based on The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database, we conducted a multi-omics analysis of METTL3 and METTL14 in HCC, including RNA-sequencing, m6ARIP-sequencing, and ribosome-sequencing profiles. We found that the expression and prognostic value of METTL3 and METTL14 are opposite in HCC. Besides, after METTL3 and METTL14 knockdown, most of the dysregulated mRNAs, signaling pathways and biological processes are distinct in HCC, which partly explains the contrary regulatory role of METTL3 and METTL14. Intriguingly, these mRNAs whose stability or translation efficiency are influenced by METTL3 or METTL14 in an m6A dependent manner, jointly regulate multiple signaling pathways and biological processes, which supports the cooperative role of METTL3 and METTL14 in catalyzing m6A modification. In conclusion, our study further clarified the contradictory role of METTL3 and METTL14 in HCC.
Project description:N6-Methyladenosine (m6A) is the most common and abundant mRNA modification that involves regulating the RNA metabolism. However, the role of m6A in regulating the ?-cell function is unclear. Methyltransferase-like 14 (METTL14) is a key component of the m6A methyltransferase complex. To define the role of m6A in regulating the ?-cell function, we generated ?-cell METTL14-specific knockout (?KO) mice by tamoxifen administration. Acute deletion of Mettl14 in ?-cells results in glucose intolerance as a result of a reduction in insulin secretion in ?-cells even though ?-cell mass is increased, which is related to increased ?-cell proliferation. To define the molecular mechanism, we performed RNA sequencing to detect the gene expression in ?KO islets. The genes responsible for endoplasmic reticulum stress, such as Ire1?, were among the top upregulated genes. Both mRNA and protein levels of IRE1? and spliced X-box protein binding 1 (sXBP-1) were increased in ?KO islets. The protein levels of proinsulin and insulin were decreased in ?KO islets. These results suggest that acute METTL14 deficiency in ?-cells induces glucose intolerance by increasing the IRE1?/sXBP-1 pathway.