YTHDF2 irCLIP-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA422164 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6367671.fastq.gz | Fastqsanger.gz | |||
| SRR6367672.fastq.gz | Fastqsanger.gz | |||
| SRR6367673.fastq.gz | Fastqsanger.gz |
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Project description:We performed irCLIP-seq to determine the mRNA targets of YTHDF2 in mouse hematopoietic progenitor cell line HPC-7
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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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Project description:RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
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Project description:RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
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Project description:RNA binding proteins (RBPs) interact with RNA targets to control an array of processes, including RNA splicing, stability, transport, and translation1-3. Dysfunctional RNA-RBP interactions contribute to pathogenesis of a plethora of human diseases1,4,5, underscoring the need for a greater understanding of the nature and dynamics of RNA-protein assemblies. The capacity to study native RNA-dependent protein assemblies in living cells, however, has been limited. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation6 was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with specific RBPs of interest. irCLIP-RNP defined landscapes of complex and multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP associations on RNA. This included cell-type-selective patterned relationships between RDAPs and primary RBPs, such as cell context-dependent reciprocal impacts of HNRNPU and NONO on each other’s RDAP landscapes. irCLIP-RNP also defined dynamic RDAP remodeling patterns in response to epidermal growth factor (EGF) and uncovered EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of mRNAs that mediate cell proliferation. The development of sequential immunoprecipitation irCLIP (RE-irCLIP) supported the same-RNA-molecule co-localization of irCLIP-RNP-identified associations. Thus, irCLIP-RNP and RE-irCLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
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Project description:The mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human acute myeloid leukemias (AML). To study the role of YTHDF2 on mRNA decay rates in leukemia, c-Kit+ cells from foetal livers of Ythdf2fl/fl; Vav-iCre (Ythdf2CKO) and Ythdf2fl/fl (Ythdf2CTL) 14.5 dpc embryos were transduced with Meis1 and Hoxa9 oncogenes and serially re-plated to generate pre-leukemic cells. Medium with 4SU was used for pre-leukemic cells labelling for 12 hours and was later replaced with 4SU-free medium (time 0). Cells were collected immediately after medium change and at 1, 3 and 9 hours for library generation. RNA from Ythdf2CKO (n=3 biological replicates) and Ythdf2CTL (n=3 biological replicates) pre-leukemic cells were used for SLAM-seq library generation.
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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 on translation regulation in leukemia, c-Kit+ cells from foetal livers of Ythdf2fl/fl; Vav-iCre (Ythdf2CKO) and Ythdf2fl/fl (Ythdf2CTL) 14.5 dpc embryos were transduced with Meis1 and Hoxa9 oncogenes and serially re-plated to generate pre-leukemic cells. RIBO-seq libraries were then prepared from Ythdf2CKO (n=3) and Ythdf2CTL (n=3) pre-leukemic cells.
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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.
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