HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [LSC and LSK]
UID: 10803
- 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 mouse HSPCs (Lin- Sca1+ Kit+ or LSK cells) and mouse leukemic stem cells (LSCs). MSI2 fusion with the catalytic domain of the Hyperactive ADAR mutant (MSI2-ADA) or with the dead catalytic mutant (MSI2-DCD) were overexpressed for 48 hours in LSKs and LSCs, followed by 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 show that despite comparable expression of the MSI2-ADA fusion protein and endogenous MSI2 in LSCs vs LSKs, MSI2 RNA binding activity (number of targets and editing frequency) in LSCs is significantly higher than that in LSKs. This elevated RNA binding activity is independent of abundancy of the target transcripts.
MSI2-ADA, MSI2-DCD fusion or MIG (empty vector) for 48 hours were overexpressed in LSK cells sorted from normal C57/BJ6 mice and LSCs sorted from quaternary MLL-AF9 Actin-DsRed mice. Cells were harvested 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 #: GSE132948Access via SRARNA Sequence reads for 18 samples.
Accession #: SRP201802Access via BioProjectAdditional information about overall initiative.
Accession #: PRJNA549511 - 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
- 29.2 (XLSX), 626.8 Kb (CSV), 27.7 (SRA)
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