Project description:RNA-binding proteins (RBPs) play pivotal roles in directing RNA fate and function. Yet the current annotation of RBPs is largely limited to proteins carrying known RNA-binding domains. To systematically reveal dynamic RNA-protein interactions, we surveyed the human proteome by a protein array-based approach and identified 671 proteins with RNA-binding activity. Among these proteins, 525 lack annotated RNA-binding domains and are enriched in transcriptional and epigenetic regulators, metabolic enzymes, and small GTPases. Using an improved CLIP (crosslinking and immunoprecipitation) method, we performed genome-wide target profiling of isocitrate dehydrogenase 1 (IDH1), a novel RBP. IDH1 binds to thousands of RNA transcripts with enriched functions in transcription and chromatin regulation, cell cycle and RNA processing. Purified IDH1, but not an oncogenic mutant, binds directly to GA- or AU-rich RNA that are also enriched in IDH1 CLIP targets. Our study provides useful resources of unconventional RBPs and IDH1-bound transcriptome, and convincingly illustrates, for the first time, the in vivo and in vitro RNA targets and binding preferences of IDH1, revealing an unanticipated complexity of RNA regulation in diverse cellular processes.
Project description:RNA-binding proteins (RBPs) play pivotal roles in directing RNA fate and function. Yet the current annotation of RBPs is largely limited to proteins carrying known RNA-binding domains. To systematically reveal dynamic RNA-protein interactions, we surveyed the human proteome by a protein array-based approach and identified 671 proteins with RNA-binding activity. Among these proteins, 525 lack annotated RNA-binding domains and are enriched in transcriptional and epigenetic regulators, metabolic enzymes, and small GTPases. Using an improved CLIP (crosslinking and immunoprecipitation) method, we performed genome-wide target profiling of isocitrate dehydrogenase 1 (IDH1), a novel RBP. IDH1 binds to thousands of RNA transcripts with enriched functions in transcription and chromatin regulation, cell cycle and RNA processing. Purified IDH1, but not an oncogenic mutant, binds directly to GA- or AU-rich RNA that are also enriched in IDH1 CLIP targets. Our study provides useful resources of unconventional RBPs and IDH1-bound transcriptome, and convincingly illustrates, for the first time, the in vivo and in vitro RNA targets and binding preferences of IDH1, revealing an unanticipated complexity of RNA regulation in diverse cellular processes.
Project description:This SuperSeries is composed of the following subset Series: GSE38356: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [RNA-seq] GSE38201: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [PAR-CLIP] GSE38355: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [protein occupancy profiling] Refer to individual Series
Project description:In the present study, we applied a quantitative MS-based strategy to characterize the proteome and phosphoproteome in HRAS- or IDH1-driven glioma cells. We describe the driving roles of the MEK and PI3K signaling pathways in RAS-NHA cells, and uncover oncogenic signaling in other pathways. We highlight the interplay between the signaling cascades and show that inhibition of MEK and PI3K reverses phosphorylation signaling patterns driven by oncogenic RAS overexpression. Applying a histone hybrid chemical labeling method and high-resolution MS, we identified significant histone methylation, acetylation, and butyrylation changes in IDH1mut-NHA cells. Our results suggest a global transcriptional repressive state, consistent with the down-regulation of the proteome, transcriptome, and the DNA hyper-methylated state in IDH1mut-NHA cells. We provide a unique resource of altered proteins, phosphosites, and histone PTMs in RAS and IDH1 mutant astrocytoma cell lines, providing insight into oncogenesis in glioma beyond the transcriptomic level.
Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129M-bM-^@M-^S141, with HEK293 cell lines stably expressing HIS/FLAG/HA-tagged ALKBH5, C17orf85, C22orf28, CAPRIN1, and ZC3H7B. We used 4-thiouridine (4SU) to enhance the crosslink and generated two PAR-CLIP cDNA libraries per protein.
Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks.
Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks.
Project description:Although, IDH1 has been recognized to play critical roles in the regulation of dynamic chromatin states, the mechanism remained largely unknown. Here, using the human erythropoiesis system, we presented an innovative perspective of metabolism independent role of nuclear IDH1. We discovered that IDH1 acted as a nuclear located chromatin-binding protein. Knockdown of IDH1 induced chromatin reorganization and subsequently aroused aberrant biological events germination on erythroid precursors in a metabolism independent manner. IDH1 knockdown induced genome-wide coordinate changes in the distribution and intensity of multiple histone marks, among which H3K79me3 was identified to be the decisive factor in chromatin state reprogramming. Integrating ChIP-seq, ATAC-seq and RNA-seq recognized SIRT1 to be the key gene affected by IDH1 deficiency. Taken together, these findings provided a novel insight for further clarifying the fundamental biological function of IDH1 which had substantial implications for an in-depth understanding of the pathogenesis of diseases with IDH1 dysfunction and accordingly the development of therapeutic strategies.