HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [LSK cells]
UID: 10802
- Description
- Summary from the GEO: " Understanding RBPs’ molecular functions as well as their cell-type specific activity requires identification of RBPs’ direct mRNA targets. However, efforts to identify RNA targets of RNA binding proteins in stem cells have been hindered by limited cell numbers. Here we adapted the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to allow for global mapping of mRNA targets of the RBP MSI2 in rare mammalian adult stem cells. We applied this method to identify MSI2 RNA binding targets in subcompartments of mouse HSPCs including long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors MPP2 and MPP4. MSI2 fusion with the catalytic domain of the Hyperactive ADAR mutant (MSI2-ADA) or with the dead catalytic mutant (MSI2-DCD) were overexpressed in sorted LSK cells, which were transplanted into mice. After 7 weeks, mouse HSPCs were sorted into 4 populations for RNA-seq. As ADAR converts A to I (G), the fusion leaves a "finger-print" where MSI2 binds. MSI2-DCD and empty vector controls were used to normalize the background editing. We were able to identify MSI2 RNA binding targets in hematopoietic stem and progenitor cells for the first time. Furthermore, we show differential RNA binding activity (by target transcripts and editing frequency) of MSI2 in different subcompartments of HSPCs.
MSI2-ADA, MSI2-DCD fusion or MIG (empty vector) were overexpressed in LSK cells sorted from normal C57/BJ6 mice for 48hrs and then transplanted into lethally irradiated C57/B6J mice. After 7 weeks, engraftment was confirmed and bone marrow cells were sorted into LT-HSCs, ST-HSCs, MPP2 and MPP4 for RNA-seq to look for A-G editing events."
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Access via GEO
GZip of CSV and XLSX Sequencing Data
Accession #: GSE132947Access via SRARNA Sequence reads for 23 samples.
Accession #: SRP201799Access via BioProjectAdditional information about overall initiative.
Accession #: PRJNA549510 - Access Restrictions
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Free to All
- Access Instructions
- The NCBI Gene Expression Omnibus, SRA, and BioProject databases provide open access to these files.
- Data Type
- Equipment Used
- Dataset Format(s)
- CSV, SRA, gzip, XLSX
- Dataset Size
- 15.5 Mb (XLSX), 731.4 Kb (CSV), 37.9 Gb (SRA)
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