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YTHDF2 irCLIP-seq

ABSTRACT: We performed irCLIP-seq to determine the mRNA targets of YTHDF2 in mouse hematopoietic progenitor cell line HPC-7

ORGANISM(S): Mus musculus

PROVIDER: GSE107953 | GEO | 2018/08/17

REPOSITORIES: GEO

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YTHDF2 irCLIP-seq

Project description:YTHDF2 irCLIP-seq

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MeRIP, RIP and RNA Sequencing of the H1299 stable cell lines which the endogenous YTHDF2 was silenced by a short hairpin RNA targeting YTHDF2 3’UTR (shYTHDF2) and then re-expressed Flag-tagged YTHDF2-WT orYTHDF2-K571R

Project description:SUMOylation affects many aspects of target proteins such as activity, stability, localization and protein-protein interactions. We have found that SUMOylation of YTHDF2 increased its binding activity with m6A-RNAs by using different experimental approaches. To confiremd this conclusion,the analysis of RIP-seq, MeRIP-seq and RNA-seq in H1299-shYTHDF2 cells re-expressing YTHDF2-WT and YTHDF2-K571R was performed. MeRIP+RIP targets showed lower binding affinities in the mutant YTHDF2-K571R when compared with YTHDF2-WT. Compared to the control group, the binding capacities of YTHDF2 to RIP targets in treated group with either 2-D08 or GA were decreased, especially to MeRIP+RIP targets. Moreover, SUMOylated YTHDF2 promoted m6A-RNAs degradation. Combined analysis of RNA-seq, RIP-seq and MeRIP-seq showed that the mRNA levels were up-regulated in shYTHDF2 stable cells re-expressing YTHDF2-K571R compared with those in re-expressing YTHDF2-WT.

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m6A-irCLIP of mouse CD4+ T cell RNA

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Rbm15-irCLIP-seq

Project description:we performed infrared crosslinking immunoprecipitation followed by sequencing (irCLIP-seq) (Zarnegar et al., 2016) for Rbm15 to directly map its binding sites on RNA. As a principle of concept, the cells were engineered to simultaneously express emGFP-Rbm15 and Xist RNA together, as Rbm15 strongly interacts with Xist A-repeat to deposit the m6A methylation downstream. Cross-linking induced truncation site (CITS or RT stops) is the main signature occurring at our irCLIP-seq datasets. RNA meta-profile plot against normalized transcripts shows that these CITSs reside across the transcript, with 2 main peaks in transcript starts and near the stop-codon regions, in agreement with the RBM15/15B binding profile in human cells (Patil et al., 2016).Motif analysis against CITSs revealed that Rbm15 binding sites prefer U-rich stretches, namely 3 or 4 consecutive Us. This is also true for crosslinking induced mutations (CIMS).

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Microarray profiling of YTHDF2 deficient mouse oocytes

Project description:To study the role of the protein YTHDF2 during female gametogenesis, its gene was conditionally deleted in oocytes. Total RNA samples from YTHDF2 deficient GV and MII oocytes (and control oocytes) were subjected to array profiling.

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Anti YTHDF2 RIP-seq to identify YTHDF2 target mRNAs in P0 mouse retinas

Project description:To identify the target mRNAs of the m6A reader protein YTHDF2, we carired out anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P0 wild type mouse retinas was pulled down by rabbit polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input and any fold change greater than 2 was considered enriched. Finally, Biological replicates of anti-YTHDF2 RIP-Seq identified 1639 transcripts. This study provides a gene list which shows mRNA binding with YTHDF2 in mouse retina.

2022-03-15 | GSE145390 | GEO

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Project description:YTHDF2 is overexpressed in a broad spectrum of human acute myeloid leukemias (AML). To study the role of YTHDF2 in leukemia, total RNA from Ythdf2CKO (n=4) and Ythdf2CTL (n=4) leukemic stem cells were used for Affymetrix global gene expression analysis.

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Anti-YTHDF1 and anti-YTHDF2 RIP-Seq to identify the target mRNAs for YTHDF1 and YTHDF2, respectively

Project description:To identify the target mRNAs of the m6A reader protein YTHDF1 and YTHDF2, we carired out anti YTHDF1 and anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P6-P8 wild type mouse cerebellum was pulled down by rabbit polyclonal anti-YTHDF1 (proteintech) or polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input and any fold change greater than 2 was considered enriched. Finally, Biological replicates of anti-YTHDF1 RIP-Seq and anti-YTHDF2 RIP-Seq identified 506 and 596 mRNAs transcripts, respectively. This study provides gene lists which shows mRNA binding with YTHDF1 and YTHDF2 in mouse cerebellum.

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YTHDF2 mediates the mRNA degradation in prostate cancer in m6A-dependent way

Project description:As the crucial m6A reader, YTHDF2 usually degrades the target mRNAs by recognizing the m6A modified sites, consequently altering m6A levels of each mRNA. In this study, we used m6A MeRIP sequencing to detect the m6A modification alterations in prostate cancer (PCa) cell line after knocking down YTHDF2 and identify how YTHDF2 promote the PCa progression by mediating the mRNA degradation in m6A-dependent way.

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