Project description:N6-methyladenosine (m6A), the most abundant messenger RNA (mRNA) modification, has emerged as a critical post-transcriptional mechanism that regulates various aspect of mRNA metabolism including pre-mRNA processing, mRNA export, mRNA stability and translation efficiency. However the genetics basis of m6A is largely unexplored. In this work, we mapped m6A-QTLs in 60 LCLs and used the resulting tens of thousands of QTLs to provide insights to m6A biology and its contribution to human complex trait.
Project description:N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A. PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down
Project description:Oxaliplatin as a first-line drug frequently causes the chemo-resistance on colorectal cancer (CRC). N6-methyladenosine (m6A) methylation has been largely acknowledged in multiple biological functions. However, the molecular mechanisms underlying the m6A methylation in modulating anticancer drug resistance in CRC are still obscure. In present study, RIP-seq was conducted to investigate the occupancy of N6-methyladenosine RNA binding protein 3 (YTHDF3) served as “readers” that can recognize m6A modification site in HCT116 cells with oxaliplatin resistance (HCT116R). Then, YTHDF3 was knockdown by siRNA in HCT116 cells with oxaliplatin resistance, and RIP-seq was further conducted to investigate m6A methylation of HCT116, HCT116R and HCT116R cells with YTHDF3 knockdown.
Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown
Project description:In type 2 diabetes, pancreatic beta-cells fail to compensate for the presence of insulin resistance in target tissues and represent a central player in the disease development. Identifying and studying innovative molecular mechanisms that lead to beta-cell failure in diabetes represent an interesting line of research and are necessary. N6-Methyladenosine (m6A) is the most abundant modification in mRNA and is found virtually in all mammals. Through m6A-profiling, we aim to characterize the dynamic RNA methylation changes in islets obtained from patients with type 2 diabetes.
Project description:We developed RADAR , a method for detecting differential methylated loci in MeRIP-seq data. RADAR uses a flexible model to accommodate increased noise due to RNA immunoprecipitation and are compatible with complex study design. RADAR enabled accurate identification of altered methylation sites in patient samples where covariates need to be accounted for. We use the m6A-profiling of mouse liver data as an example data set to benchmart the performance of RADAR and a few alternative methods.