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SRP109284
Homo sapiens
6 Downloadable Samples
Illumina HiSeq 2500
Description
Immunodeficient mouse models have been valuable for studies of human hematopoiesis, but high-fidelity recapitulation of erythropoiesis in most xenograft recipients remains elusive. Recently developed immunodeficient and Kit mutant mice, however, have provided a suitable background to achieve higher-level human erythropoiesis after long-term hematopoietic engraftment. While there has been some characterization of human erythropoiesis in these models, a comprehensive analysis of various developmental stages has not yet been reported. Here, we have utilized cell surface phenotypes, morphologic analyses, and molecular studies to fully characterize human erythropoiesis from multiple developmental stages in immunodeficient and Kit mutant mouse models following long-term hematopoietic stem and progenitor cell engraftment. We show that human erythropoiesis in such models demonstrates complete maturation and enucleation, as well as developmentally appropriate globin gene expression. These results provide a framework for future studies to utilize this model system for interrogating disorders affecting human erythropoiesis and for developing improved therapeutic approaches. Overall design: (mRNA-seq) RNA-seq of human CD235a+ cells isolated 14-16 weeks post-implantation from mouse bone marrow were performed for three biological replicates each of mice xenograted with adult bone marrow-derived human CD34+ cells and cord blood-derived CD34+ cells.
Publication Title
Developmentally-faithful and effective human erythropoiesis in immunodeficient and Kit mutant mice.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 25 Downloadable Samples
- IlluminaHiSeq2500, IlluminaHiSeq2000
Description
Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We have 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 (Baltz and Munschauer et al. 2012). Our current work focuses on streamlining and extending protein occupancy profiling on poly(A)-RNA. Our objectives are to identify previously unknown protein-bound transcripts and, more importantly, to assess global and local differences in protein occupancy across different biological conditions. To this end, we have implemented poppi, the first pipeline for differential analysis of protein occupancy profiles. We have applied our analysis pipeline to pinpoint changes in occupancy profiles of MCF7 cells against already published HEK293 cells [GSE38157]. Overall design: We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled MCF7 cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced.
Publication Title
Differential protein occupancy profiling of the mRNA transcriptome.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
- IlluminaHiSeq2500
Description
RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5'' to 3'' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3'' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5'' to 3'' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3'' UTRs. Overall design: Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were used to determine the protein-RNA interaction sites of RNA helicases MOV10 and UPF1 as well as MOV10 inactive variants using PAR-CLIP in combination with next generation sequencing. mRNA half-life changes of MOV10-targeted mRNA were determined by measuring mRNA half-lives by mRNA sequencing of mock and MOV10-depleted HEK293 cells.
Publication Title
MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28 binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knock down, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets that function in translation, mRNA splicing and cell cycle control. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts. Overall design: We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA binding domains of LIN28B in the human transcriptome at nucleotide resolution.
Publication Title
Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 4 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
FMRP targets distinct mRNA sequence elements to regulate protein expression.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 4 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Fragile-X Syndrome (FXS) is a multi-organ disease leading to mental retardation, macro-orchidism in males, and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASD). FXS is typically caused by the loss of FRAGILE X-MENTAL RETARDATION 1 (FMR1) expression, which encodes for the RNA-binding protein (RBP), FMR1 (or FMRP). We report the discovery of the RNA recognition elements (RREs), binding sites, and mRNA targets for wild-type and I304N mutant FMRP isoforms as well as its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine target mRNA association with FMRP. Among highly-enriched targets, we identified many genes involved in ASD and demonstrate that FMRP can affect their protein levels in cell culture, mice, and human brain. Unexpectedly, we discovered that these targets are also dysregulated in Fmr1-/- mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that FMRP targets shared signaling pathways across different cellular contexts. As it is become increasingly appreciated that signaling pathways are important to FXS and ASD, our results here provide an invaluable molecular guide towards the pursuit of novel therapeutic targets for these devastating neurological disorders.
Publication Title
FMRP targets distinct mRNA sequence elements to regulate protein expression.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 16 Downloadable Samples
- Illumina HiSeq 2500
Description
Thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen lncRNAs. One specific lncRNA, Non-coding RNA Activated by DNA Damage (NORAD), has recently been shown by genetic deletion to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here, we combine RNA antisense purification (RAP) and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX (an emerging component of the DNA-damage response) and encodes the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex, which we term NORAD-Activated Ribonucleoprotein Complex 1 (NARC1), containing known suppressors of genomic instability: topoisomerase I (TOP1), ALYREF and the PRPF19/CDC5L complex. Cells depleted of NORAD or RBMX display an increased frequency of chromosome segregation errors, reduced replication-fork velocity and altered cell cycle progression phenotypes that are mechanistically linked to TOP1 and PRPF19/CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function and that NORAD is required for the assembly of a previously unknown topoisomerase complex (NARC1) that contributes to maintaining genomic stability. Moreover, we uncover a novel function for lncRNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex. Overall design: We examined gene expression changes and alternative splicing events in wildtype and NORAD depleted cells using RNA sequencing.
Publication Title
The NORAD lncRNA assembles a topoisomerase complex critical for genome stability.
Sample Metadata Fields
Cell line, Subject, Time
View Samples- Homo sapiens
- 14 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
To assess whether the transcripts identified by PAR-CLIP are regulated by the RNA-binding protein (RBP) Quaking (QKI), we analyzed the mRNA levels of mock-transfected and QKI-specific siRNA-transfected cells with microarrays. Transcripts crosslinked to QKI were significantly upregulated upon siRNA transfection, indicating that QKI negatively regulates bound mRNAs (Figure 3H of PMID 20371350), consistent with previous reports of QKI being a repressor.
Publication Title
Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
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. Overall design: We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced
Publication Title
The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
Sample Metadata Fields
Treatment, Subject
View Samples