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Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [RNA-Seq]

ABSTRACT: Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78508 | GEO | 2016/04/07

SECONDARY ACCESSION(S): PRJNA313161

REPOSITORIES: GEO

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Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [eCLIP-Seq]

Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis

2016-04-07 | E-GEOD-78507 | biostudies-arrayexpress

Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [RNA-Seq]

2016-04-07 | E-GEOD-78508 | biostudies-arrayexpress

Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [eCLIP-Seq]

2016-04-07 | GSE78507 | GEO

IMP1 and IMP2 have different RNA clients and functional programs in mouse embryo fibroblasts

Project description:Different from most publication to date, in this study we find IMP1 and IMP2, two closely related RNA binding proteins, have different clients in MEF cells

2019-12-22 | GSE133242 | GEO

Enhanced CLIP-seq analysis of PTBP1 and IGF2BP2 binding targets

Project description:To investigate the binding targets of PTBP1 (PTB) and IGF2BP2 (IMP2) in glioma cells, we carried out an unbiased analysis of transcripts bound by PTBP1 and IGF2BP2 using enhanced crosslinking immunoprecipitation followed by high-throughput sequencing (eCLIP) in A172 glioma cells. We repeated the eCLIP method twice in each experiment, and found that the ratio of PTBP1 and IGF2BP2 binding mRNAs overlap was very high.

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RNA seq from ADAR fused IMP mouse liver transformants TRIBE

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Expression data from human induced pluripotent stem cell-derived teratomas and embryoid bodies

Project description:The tumorigenicity of human pluripotent stem cells (hPSCs) is a major safety concern for their application in regenerative medicine. Here we identify the tight-junction protein Claudin-6 as a specific cell surface marker of hPSCs that can be used to selectively remove Claudin-6-positive cells from mixed cultures. We show that Claudin-6 is absent in adult tissues but highly expressed in undifferentiated cells, where it is dispensable for hPSC survival and self-renewal. We use three different strategies to remove Claudin-6-positive cells from mixed populations: an antibody against Claudin-6; a cytotoxin-conjugated antibody that selectively targets undifferentiated cells; and clostridium perfringens enterotoxin, a toxin that binds several Claudins, including Claudin-6, and efficiently kills undifferentiated cells, thus eliminating the tumorigenic potential of hPSC-containing cultures. This work provides a proof of concept for the use of Claudin-6 to eliminate residual undifferentiated hPSCs from culture, highlighting a strategy that may increase the safety of hPSC-based cell therapies. total RNA was isolated from teratomas or from embryoid bodies differentiated from human induced pluripotent stem cells

2013-06-01 | E-GEOD-38088 | biostudies-arrayexpress

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m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

Project description:Human neurodevelopment requires differentiating neurons to establish large networks of connections in a highly stereotyped manner. Neuronal differentiation in particular, requires RNA-binding proteins to spatiotemporally regulate thousands of different mRNAs. Yet, how these proteins precisely relate to neuronal development and coordinate the expression of functionally coherent genes in a cell type specific manner is only partially understood. To address this, we sought to understand how the paradigmatic RNA-binding protein IMP1/IGF2BP1, an essential developmental factor, selects and regulates its RNA targets transcriptome-wide during the differentiation of human neurons. We used a combination of systemic and molecular analyses to show that IMP1 directly binds to and regulates the expression of a large set of mRNAs that govern microtubule assembly, an essential process and a key driver of neuronal differentiation. We also show that m6A methylation during the transition from neural precursors to neurons drives both the selection of IMP1 mRNA targets and their translation potential. Our findings establish m6A methylation as a key mechanism coordinating the regulatory action of IMP1 on human neuronal architecture.

2024-06-10 | PXD034341 | Pride

IMP2 axonal localization, RNA interactome, and function in development of axon trajectories

Project description:RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurologic disease. Developing axons provide a well suited model to study RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal its requirement for normal axon pathfinding during vertebrate development. CLIP-seq

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