Project description:N6-methyladenosine (m6A) modification, as the most abundant internal methylation of eukaryotic RNA transcripts, is critically implicated in RNA processing, decay, transport and translation. Here we showed that KIAA1429, the largest known component in the m6A methyltransferase complex, was considerably upregulated in hepatocellular carcinoma (HCC) tissues. High expression of KIAA1429 was significantly associated with the malignant clinical features and the poor prognosis of HCC patients. Silencing KIAA1429 suppressed the cell proliferation and metastasis in vitro and in vivo. Integrated MeRIP-seq, RIP-seq and RNA-seq data identified GATA3 as the direct downstream target of KIAA1429-mediated m6A modification. KIAA1429 knockdown markedly impaired the m6A levels of GATA3 mRNA and increased the expression of GATA3. Mechanistically, KIAA1429 induced the m6A methylation on 3’ UTR of GATA3 pre-mRNA, leading to the separation of RNA-binding protein HuR and the degradation of GATA3 pre-mRNA, which was followed by the downregulation of GATA3. Strikingly, a long noncoding RNA (lncRNA) GATA3-AS, transcribed from the antisense strand of GATA3 gene, functioned as a cis-acting element for the preferential interaction of KIAA1429 with GATA3 pre-mRNA. Accordingly, we found that the tumor growth and metastasis driven by KIAA1429 or GATA3-AS were mediated by GATA3. Patients with low KIAA1429 and high GATA3 expressions showed greatly better poor overall survival and disease-free survival. In conclusion, our study proposed a complex KIAA1429-GATA3 regulatory model based on m6A modification and provided insights into the epi-transcriptomic dysregulation in hepatocarcinogenesis and metastasis.
Project description:The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-Activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL11 mRNA, inducing autocrine of IL11 and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for anti-metastatic therapies. To identify mRNA species bound by lncRNA-ATB, we performed an RIP to pull down endogenous mRNAs associated with the lncRNA-ATB and sequenced the retrieved RNA.
Project description:Large-scale analyses have revealed that 60-90% of the mammalian genome is transcriptionally active. Because <2% of sequences have protein-coding potential, why so much cellular energy is expended on RNA synthesis is a major question in the post-genomic era. The hypothesis that RNA may serve as recruiting platforms for chromatin modifiers has gained ground with discoveries linking long ncRNAs, such as RepA, Xist, and Tsix, to locus-specific targeting of Polycomb proteins to the X-chromosome. Long ncRNAs have also been associated with Polycomb proteins at human HOX loci. Here, we ask if RNA targeting may be a general feature of regulation for mouse Polycomb repressive complex 2 (PRC2). We develop a method to capture the 'PRC2 transcriptome' by combining RNA immunoprecipitation (RIP) with deep sequencing (seq). RIP-seq of mouse embryonic stem cells identifies >3000 RNAs in the PRC2 transcriptome. Approximately 14% of RNAs originate from previously described PRC2-binding sites and many are promoter-associated. The transcriptome includes a large number of unannotated noncoding and antisense RNAs, with the X-chromosome exhibiting a high density of PRC2 RNAs. Imprinted genes and other disease genes, including those involved in cancer, are also well-represented. Functional validation of select candidates confirms RNA-PRC2 interactions in vivo and recruitment of Polycomb proteins in cis. Thus, RNA cofactors may be one general mechanism, among others, for targeting mammalian PRC2. Given PRC2's essential roles during stem cell renewal, development, and cancer, the PRC2 transcriptome described herein will provide a valuable resource for regenerative medicine and cancer biology. Identification and characterization of RNAs associated with PRC2 complex in mouse embryoinc stem cells
Project description:We identified the mRNA targets of the insulin-like growth factor-2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) by RNA immunoprecipitation and sequencing (RIP-seq). HEK293T cells transfected with Flag-tagged IGF2BP1/2/3 plasmids were expanded and UV-crosslinked before harvest. We performed RIP of individual IGF2BP using anti-Flag antibody from nuclear extractions, and identified the associated mRNAs by next generation sequencing. More than 5000 transcripts, including protein coding and non-coding transcripts, were identified from each RIP-seq sample.
Project description:To identify the directly bound transcripts of Flag antibody (the backgroud control for our METTL16 RIP-seq), RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T. Briefly, HEK293T cells were infected with pmiRNA1-empty vector. Only the GFP-positive cells were used for study and expanded in DMEM medium.
Project description:Large-scale analyses have revealed that 60-90% of the mammalian genome is transcriptionally active. Because <2% of sequences have protein-coding potential, why so much cellular energy is expended on RNA synthesis is a major question in the post-genomic era. The hypothesis that RNA may serve as recruiting platforms for chromatin modifiers has gained ground with discoveries linking long ncRNAs, such as RepA, Xist, and Tsix, to locus-specific targeting of Polycomb proteins to the X-chromosome. Long ncRNAs have also been associated with Polycomb proteins at human HOX loci. Here, we ask if RNA targeting may be a general feature of regulation for mouse Polycomb repressive complex 2 (PRC2). We develop a method to capture the 'PRC2 transcriptome' by combining RNA immunoprecipitation (RIP) with deep sequencing (seq). RIP-seq of mouse embryonic stem cells identifies >3000 RNAs in the PRC2 transcriptome. Approximately 14% of RNAs originate from previously described PRC2-binding sites and many are promoter-associated. The transcriptome includes a large number of unannotated noncoding and antisense RNAs, with the X-chromosome exhibiting a high density of PRC2 RNAs. Imprinted genes and other disease genes, including those involved in cancer, are also well-represented. Functional validation of select candidates confirms RNA-PRC2 interactions in vivo and recruitment of Polycomb proteins in cis. Thus, RNA cofactors may be one general mechanism, among others, for targeting mammalian PRC2. Given PRC2's essential roles during stem cell renewal, development, and cancer, the PRC2 transcriptome described herein will provide a valuable resource for regenerative medicine and cancer biology.