Wilms' Tumor 1-associating Protein Research Articles

WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).

MiR-550-1 functions as a tumor suppressor in acute myeloid leukemia via the hippo signaling pathway.

MicroRNAs (miRNAs) and N6-methyladenosine (m6A) are known to serve as key regulators of acute myeloid leukemia (AML). Our previous microarray analysis indicated miR-550-1 was significantly downregulated in AML. The specific biological roles of miR-550-1 and its indirect interactions and regulation of m6A in AML, however, remain poorly understood. At the present study, we found that miR-550-1 was significantly down-regulated in primary AML samples from human patients, likely owing to hypermethylation of the associated CpG islands. When miR-550-1 expression was induced, it impaired AML cell proliferation both in vitro and in vivo, thus suppressing tumor development. When ectopically expressed, miR-550-1 drove the G0/1 cell cycle phase arrest, differentiation, and apoptotic death of affected cells. We confirmed mechanistically that WW-domain containing transcription regulator-1 (WWTR1) gene was a downstream target of miR-550-1. Moreover, we also identified Wilms tumor 1-associated protein (WTAP), a vital component of the m6A methyltransferase complex, as a target of miR-550-1. These data indicated that miR-550-1 might mediate a decrease in m6A levels via targeting WTAP, which led to a further reduction in WWTR1 stability. Using gain- and loss-of-function approaches, we were able to determine that miR-550-1 disrupted the proliferation and tumorigenesis of AML cells at least in part via the direct targeting of WWTR1. Taken together, our results provide direct evidence that miR-550-1 acts as a tumor suppressor in the context of AML pathogenesis, suggesting that efforts to bolster miR-550-1 expression in AML patients may thus be a viable clinical strategy to improve patient outcomes.

WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1

BackgroundN6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer.MethodsWe determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay.ResultsWe demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1.ConclusionWe have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment.

Physiological functions of Wilms' tumor 1-associating protein and its role in tumourigenesis.

The Wilms' tumor-associated gene WT1 encodes a tumor suppressor gene, which is implicated in renal differentiation and development of adult urogenital system. Wilms' tumor 1-associating protein (WTAP) is initially identified as a nuclear protein that specifically interacts with WT1 in both in vitro and in vivo assays. WTAP is ubiquitously expressed in different tissues and various growth periods, and its expression is involved in cell cycle, RNA splicing and stabilization, N6-methyladenosine RNA modification, cell proliferation, and apoptosis as well as embryonic development. In the present review, we aimed to summarize the functions of WTAP in various physiological and pathological processes, in particular with regard to the current knowledge about the role of WTAP in tumorigenesis of different cancers.

The role of N6-methyladenosine RNA methylation in the heat stress response of sheep (Ovis aries).

With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

Relationship between the genetic expression of WTAP and bladder cancer and patient prognosis.

The relationship between the gene expression of Wilms tumor 1-associated protein (WTAP) and bladder cancer was investigated to study the effect of its expression on patient prognosis. Sixty-two fresh specimens of bladder transitional cell cancer tissues were collected as the bladder cancer group, while 20 normal bladder mucosa specimens comprised the control group. Hematoxylin and eosin staining was conducted to detect the pathological differences between the groups and the immunohistochemistry was used to test the expression levels of WTAP in the tissues. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of WTAP. Moreover, western blot analysis was used to examine the WTAP expression levels. At the same time, Cox regression multi-factor survival analysis was conducted for the related factors to the prognoses of bladder cancer patients. The structures of cells in the bladder cancer group were destroyed as was evident by the shrunken cell nuclei, while the tissues in the control group were intact. WTAP expression in the bladder cancer group was significantly increased compared with that in the control group. A small number of mRNAs and proteins were higher in the bladder cancer group than that in the control group. The differences in WTAP expression between the bladder cancer and control groups were statistically significant (P<0.05). There were obvious differences in the postoperative recurrence risk between the patients with a negative WTAP protein expression and those with a positive one (P<0.05). In conclusion, WTAP may play an important role in the occurrence and development of bladder cancer and can be considered as a potential target for bladder cancer treatment, providing a new basis for clinical diagnoses.

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis.

Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear. N6-Methyladenosine (m6A) posttranscriptional RNA modification, whose methylation and demethylation are performed by respective enzyme molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N6-adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like 3 (METTL3), and METTL14 positively controls adipogenesis by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistent with this, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through the WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders.

METTL3 regulates WTAP protein homeostasis

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex.

Abstract 4442: Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification

Abstract Purpose: Wilms' tumor 1-associating protein (WTAP) which is known for its role in RNA methylation machinery, is highly upregulated in the necrotic region of glioblastoma multiforme (GBM). The highly heterogenic nature of GBM is associated with the necrotic region within the brain, where many types of cells such as the stromal cell, immune cells, and cancer stem cells co-localize. The complex cellular network within the necrotic region is believed to cause the resistance of the conventional chemotherapy and radiotherapy. Here we attempted to identify the role of WTAP in GBM and what kind of RNA modification is associated with the proliferation and stemness in GBM. Improvement of our understanding about post-transcriptional regulation in GBM will allow us to find out the key oncogenic switch in GBM, and development of therapeutics targeting this mechanism will provide better survival benefits. Methods: 23 Patient-derived cell lines, two established GBM cell lines, and astrocyte was arrayed to find out the correlation between WTAP and cancer stem cells. shWTAP and WTAP overexpression vector were infected into patient-derived GBM cancer stem cell lines to test the effect of WTAP. Western blot and polymerase chain reaction analysis was performed to find out the change in genes which are related to cell proliferation and stemness. Limiting dilution assay was performed to validate the function of WTAP in sphere-forming ability. RNA sequencing was performed on shWTAP infected patient-derived cancer stem cell to identify the type of RNA modification made by knocking down WTAP. Results: Knocking down of WTAP using shWTAP in patient-derived cancer stem cell lines, we were able to see the decrease in cell proliferation and stemness-related genes while overexpressing WTAP in these cells slightly increased cell proliferation and stemness. WTAP knockdown cells had a significant decrease in sphere-forming ability. RNA sequencing of shWTAP cells has revealed the increase in intron retention and the decrease in exon skipping, which shows that WTAP has a role in decreasing intron retention while increasing exon skipping. Conclusion: We have revealed that WTAP has an important role in increasing proliferation and maintaining stemness in GBM. We have also identified that WTAP decreases intron retention and increasing exon skipping. Citation Format: Sae Whan Park, Young Taek Oh, Jong Bae Park, Jong Heon Kim. Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4442.

N6-methyladenosine mediates the cellular proliferation and apoptosis via microRNAs in arsenite-transformed cells

N6-methyladenosine (m6A) modification is implicated to play an important role in cellular biological processes, but its regulatory mechanisms in arsenite-induced carcinogenesis are largely unknown. Here, human bronchial epithelial (HBE) cells were chronically treated with 2.5 μM arsenite sodium (NaAsO2) for about 13 weeks and these cells were identified with malignant phenotype which was demonstrated by increased levels of cellular proliferation, percentages of plate colony formation and soft agar clone formation, and high potential of resistance to apoptotic induction. Our results firstly demonstrated that m6A modification on RNA was significantly increased in arsenite-transformed cells and this modification may be synergistically regulated by methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor 1-associated protein (WTAP) and Fat mass and obesity-associated protein (FTO). In addition, knocking down of METTL3 in arsenite-transformed cells can dramatically reverse the malignant phenotype, which was manifested by lower percentages of clone and colony formation as well as higher rates of apoptotic induction. Given the critical roles of miRNAs in cellular proliferation and apoptosis, miRNAs regulated by m6A in arsenite-transformed cells were analyzed by Venn diagram and KEGG pathway in this study. The results showed that these m6A-mediated miRNAs can regulate pathways which are closely associated with cellular proliferation and apoptosis, implicating that these miRNAs may be the critical bridge by which m6A mediates dysregulation of cell survival and apoptosis in arsenite-transformed cells. Taken together, our results firstly demonstrated the significant role of m6A in the prevention of tumor occurrence and progression induced by arsenite.

Abstract 3343: RNA demethylase alkbh5 supports breast cancer growth and progression

Abstract N6-methyladenosine (m6A) is one of the most prevalent internal modifications in eukaryotic messenger RNA. This dynamic and reversible modification is installed by methyltransferase complex consisting of three subunits: Methyltransferase-like protein 3 (METTL3), Methyltransferase-like protein 14 (METTL14) and Wilms tumor 1-associating protein (WTAP), and erased by two independent demethylases, Fat mass and obesity associated protein (FTO) and AlkB homolog 5 (ALKBH5). RNA demthylase ALKBH5 has been reported to play vital roles in several biological processes. However, very little is known about the role of ALKBH5 in cancer in general and breast tumorigenesis in particular. In this study, we report that ALKBH5 promotes breast cancer growth and progression. Using in vitro and in vivo models, we show that silencing of ALKBH5 inhibits breast cancer growth and invasion. Importantly, our studies reveal that ALKBH5 mediates its pro-tumorigenic function by regulating several microtubule associated genes including KIF2C, PLK1, MAPRE1 and RCC2 that are critical for spindle formation or assembly during mitotic progression. Interestingly, ALKBH5 target genes KIF2C and MAPRE1 are highly expressed in breast cancer patients and their higher expression is strongly correlated with lower survival of breast cancer patients. We believe that an optimal level of RNA methylation of key target genes is critical for normal cell cycle progression and any alteration in the levels of RNA methylation may alter activity or expression of these genes resulting in unabated cell proliferation. Citation Format: Vijay Kumar Eedunuri, Panneerdoss Subbarayalu, Subhapriya Rajamanickam, Abdelfattah Nourhan, Benjamin C. Onyeagucha, Santosh Timilsina, Pooja Yadav, Manjeet K. Rao. RNA demethylase alkbh5 supports breast cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2017-3343

RNA methylation in lymphoid malignancies

N 6 -methyl-adenosine (m 6 A) modification is the most abundant internal modification in mammalian RNA and plays an important role in gene expression mechanisms such as mRNA splicing, nuclear export, transcript stability and translational efficiency. The extent and the significance of m 6 A modifications in pathological conditions, especially hematological malignancies such as lymphomas are not known. In this study, the global m 6 A methylation by an immunocapture method in normal B cells (GMO 6990), and in the B cell neoplastic cells such as diffuse large B cell lymphoma (DB), Burkitt’s lymphoma (Raji) and a relapsed pre-B cell acute lymphoblastic leukemia (ALL) (NALM6) cells are discussed. Variable mRNA expression of RNA methylases such as methyltransferase like 3 (METTL3) and Wilms tumor 1 associated protein (WTAP) that methylates internal adenosine residues in mRNA was observed in normal and lymphoma cells as quantified by qRT-PCR. Further, an mRNA demethylase, Fat mass and obesity associated enzyme (FTO) mRNA expression was found to be high in lymphoma cells compared to normal B cells. A similar trend was also observed between normal and ALL patients at diagnosis. Overall, our studies suggest that altered RNA methylation and enzymes controlling this process may be of significance in the pathology of lymphoid malignancies.

WT1-associated protein is a novel prognostic factor in pancreatic ductal adenocarcinoma.

Although Wilms tumor 1 (WT1)-associated protein (WTAP) was initially found to be a specific WT1-binding protein, it has increasingly attracted attention because of its oncogenic role in various types of malignancies, including cholangiocarcinoma, glioblastoma and acute myeloid leukemia. However, the clinical impact of WTAP on pancreatic ductal adenocarcinoma (PDAC) is still unknown. A total of 145 patients who underwent surgical treatment from 2004 to 2008 were enrolled in the present study. The cytoplasmic and nuclear expression of WTAP in tumor and adjacent normal tissues was examined by immunohistochemical analysis in order to investigate the relationship between WTAP and the clinicopathological factors and prognosis of patients with PDAC. The nuclear and cytoplasmic expression of WTAP in tumor tissues was significantly higher compared with non-tumor tissues (P<0.001). High expression of WTAP in the nucleus was significantly associated with gender (P=0.010) and tumor stage (P=0.020), while high expression of WTAP in the cytoplasm was significantly associated with gender (P=0.018), histological grade (P=0.047) and perineural invasion (P=0.028). In addition, a univariate analysis revealed that high nuclear expression of WTAP in tumor tissues was significantly associated with poor overall survival (P<0.001), as well as several clinicopathological variables, including gender and N stage. In a multivariate Cox regression analysis, nuclear WTAP expression was identified as an independent prognostic indicator for PDAC (relative risk, 1.855; 95% confidence interval, 1.033–3.333; P=0.039). The results of the present study indicated that high nuclear expression of WTAP is a valuable molecular biomarker of a poor prognosis among patients with PDAC.

Overexpression of miR-29a reduces the oncogenic properties of glioblastoma stem cells by downregulating Quaking gene isoform 6.

Glioblastoma is the most common type of malignant primary brain tumor and has high recurrence and lethality rates. Glioblastoma stem cells (GSCs), a subpopulation of glioblastoma cells, may promote rapid tumor recurrence and therapy resistance. Because altered microRNA (miR) expression in GSCs may lead to glioblastoma progression, we assessed the effects of miR-29a expression on the oncogenic behavior of GSCs. MiR-29a expression was lower in GSCs than non-GSCs, and overexpression of miR-29a in GSCs inhibited cell proliferation, migration and invasion, but promoted apoptosis. MiR-29a directly inhibited the expression of Quaking gene isoform 6 (QKI-6) by binding to its 3′-UTR, and thus inhibited GSC malignant behavior. In addition, Wilms’ tumor 1-associating protein (WTAP) was identified as a downstream target of QKI-6. Overexpression of miR-29a in GSCs inhibited expression of WTAP and suppressed both phosphoinositide 3-kinase/AKT and extracellular signal-related kinase pathways by downregulating QKI-6, thereby inhibiting cell proliferation, migration, and invasion but promoting apoptosis. We have characterized a novel miR-29a/QKI-6/WTAP axis in GSCs, which may provide theoretical support for the treatment of glioblastoma with miR-29a agomirs.

Identifying the role of Wilms tumor 1 associated protein in cancer prediction using integrative genomic analyses

The Wilms tumor suppressor, WT1 was first identified due to its essential role in the normal development of the human genitourinary system. Wilms tumor 1 associated protein (WTAP) was subsequently revealed to interact with WT1 using yeast two-hybrid screening. The present study identified 44 complete WTAP genes in the genomes of vertebrates, including fish, amphibians, birds and mammals. The vertebrate WTAP proteins clustered into the primate, rodent and teleost lineages using phylogenetic tree analysis. From 1,347 available SNPs in the human WTAP gene, 19 were identified to cause missense mutations. WTAP was expressed in bladder, blood, brain, breast, colorectal, esophagus, eye, head and neck, lung, ovarian, prostate, skin and soft tissue cancers. A total of 17 out of 328 microarrays demonstrated an association between WTAP gene expression and cancer prognosis. However, the association between WTAP gene expression and prognosis varied in distinct types of cancer, and even in identical types of cancer from separate microarray databases. By searching the Catalogue of Somatic Mutations in Cancer database, 65 somatic mutations were identified in the human WTAP gene from the cancer tissue samples. These results suggest that the function of WTAP in tumor formation may be multidimensional. Furthermore, signal transducer and activator of transcription 1, forkhead box protein O1, interferon regulatory factor 1, glucocorticoid receptor and peroxisome proliferator-activated receptor γ transcription factor binding sites were identified in the upstream (promoter) region of the human WTAP gene, suggesting that these transcription factors may be involved in WTAP functions in tumor formation.

WTAP Expression Predicts Poor Prognosis in Malignant Glioma Patients.

Wilms' tumor 1-associating protein (WTAP) interacts with the Wilms' tumor 1 gene. Although originally classified as a tumor suppressor, WTAP was later found to be over-expressed in glioblastoma which is regarded as a grade IV astrocytoma. However, the expression in other glioma grades and the relationship between WTAP expression and the prognosis of glioma patients are still unknown. In this study, we investigated WTAP expression in 169 different types of glioma cases using western blot analysis and immunohistochemistry assay. Further, we evaluated the association of WTAP expression with clinicopathological characteristics using chi-square test and Spearman's correlation test. We used univariate and multivariate Cox regression analyses to evaluate the independency of diferent WTAP expression. Then, the survival curves were calculated using the Kaplan-Meier method. Results showed that WTAP was over-expressed in glioma tissues, and the expression was closely correlated with glioma grade. Moreover, high WTAP expression was correlated with poor postoperative survival in glioma patients. WTAP may serve as a novel prognostic marker.

RNA epigenetic modification: N6-methyladenosine.

N(6)-methyladenosine (m(6)A) is one of the most prevalent internal modifications in eukaryotic messenger RNA. The dynamic and reversible modification is installed by methyltransferase complex charactered three subunits: METTL3 (Methyltransferase-like protein 3), METTL14 (Methyltransferase-like protein 14) and WTAP (Wilms tumor 1-associating protein), and erased by two independent demethylases, FTO (Fat mass and obesity associated protein) and ALKBH5 (AlkB homolog 5), in an α-ketoglutarate (α-KG)- and Fe(II)-dependent manner. m(6)A plays funtions in controlling RNA metabolism through the recognition by m(6)A reader proteins, the YTH domain family proteins and HNRNPA2B1 (Heterogeneous nuclear ribonucleoproteins A2B1) . In this review, we summarized distributive features and vital roles of m(6)A and its associated proteins in RNA metabolisms and biological significance, which will help us better understand this new exciting emerging epitranscriptome research field.

Carbonic Anhydrase IV Inhibits Colon Cancer Development by Inhibiting WNT Signaling Pathway Through Targeting WTAP-WT1-TBL1 Axis

Using Methylated DNA Immunoprecipitation (MeDIP) combined with microarray assay, Carbonic Anhydrase IV (CA4) was identified to be preferentially methylated in colorectal cancer (CRC). CA4 is a member of carbonic anhydrases family which catalyzes the reversible hydration of carbondioxide. However, the functional role of CA4 in CRC is still unclear. CA4 promoter methylation status was evaluated by bisulfite genomic sequencing. The biological functions of CA4 were determined in vitro cell viability, colony formation, cell apoptosis, and cell cycle assays and in vivo tumorigenicity assay. The cooperators of CA4 were identified by co-immunoprecipitation assay, mass spectrometry, promoter luciferase reporter assay, chromatin immunoprecipitation assay and gel shift assay. Clinical application of CA4 methylation was assessed in 115 primary CRC tissues. We first screened the expression status of CA4 in 9 CRC cell lines and primary CRC tissues. CA4 was silenced in all 9 cell lines and the mean protein expression level of CA4 was significantly lower in CRC tissues as compared to their adjacent normal tissues (P<0.01). Silence of CA4 was mediated by promoter methylation. Methylation status of CA4 was detected in 75.7% (87/115) of CRC patients. Multivariate analysis and recurrence curve revealed that patients with CA4 methylation had significantly higher opportunities to suffer from cancer recurrence (P < .05). Re-expression of CA4 in CRC cell lines suppressed cell viability (P < .001) and colony formation (P < .01), and caused cell cycle arrest in G1 phase (P < .05). CA4 also induced cell apoptosis (P < .01), concomitant with enhanced protein expression of pro-apoptotic factors cleaved caspase3, caspase7, caspase8 and PARP. The in vivo growth of CRC cells in nude mice was also markedly inhibited by stable transfection with vector harboring CA4 (P < .05). We further tested the molecular mechanisms of CA4 in CRC as a tumor suppressor. Co-immunoprecipitation assay revealed that Wilms tumor 1-associating protein (WTAP) is directly interacted with CA4. Transfection of CA4 in CRC cell lines induced WTAP protein degradation through poly-ubiquitination. Moreover, CA4 promoted transcriptional activity of Wilms tumor 1 (WT1), a negative regulator of Wnt pathway, leading to induction of transducing β-like protein 1(TBL1). TBL1 induced β-catenin degradation through a non-phosphorylated way which finally inhibit Wnt signaling pathway activity. Epigenetic inactivation of CA4 is a common event in CRC which may play a pivotal role as a novel tumor suppressor in CRC carcinogenesis. CA4 inhibit Wnt signaling pathway by targeting WTAP-WT1-TBL1 axis. Detection of CA4 methylation may serve as an independent biomarker for the recurrence of CRC.

The Drosophila Wilms׳ Tumor 1-Associating Protein (WTAP) homolog is required for eye development

Sine Oculis (So), the founding member of the SIX family of homeobox transcription factors, binds to sequence specific DNA elements and regulates transcription of downstream target genes. It does so, in part, through the formation of distinct biochemical complexes with Eyes Absent (Eya) and Groucho (Gro). While these complexes play significant roles during development, they do not account for all So-dependent activities in Drosophila. It is thought that additional So-containing complexes make important contributions as well. This contention is supported by the identification of nearly two-dozen additional proteins that complex with So. However, very little is known about the roles that these additional complexes play in development. In this report we have used yeast two-hybrid screens and co-immunoprecipitation assays from Kc167 cells to identify a biochemical complex consisting of So and Fl(2)d, the Drosophila homolog of human Wilms׳ Tumor 1-Associating Protein (WTAP). We show that Fl(2)d protein is distributed throughout the entire eye-antennal imaginal disc and that loss-of-function mutations lead to perturbations in retinal development. The eye defects are manifested behind the morphogenetic furrow and result in part from increased levels of the pan-neuronal RNA binding protein Embryonic Lethal Abnormal Vision (Elav) and the RUNX class transcription factor Lozenge (Lz). We also provide evidence that So and Fl(2)d interact genetically in the developing eye. Wilms׳ tumor-1 (WT1), a binding partner of WTAP, is required for normal eye formation in mammals and loss-of-function mutations are associated with some versions of retinoblastoma. In contrast, WTAP and its homologs have not been implicated in eye development. To our knowledge, the results presented in this report are the first description of a role for WTAP in the retina of any seeing animal.

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

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.