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HyperTRIBE Analysis for DDX41 in human leukemia cells

ABSTRACT: In HyperTRIBE analysis, a hyperactive mutant of the catalytic domain of ADAR enzyme is fused to an RNA-binding protein of interest in order to identify RNAs that are bound by the protein interest through RNA-sequencing (Rahman et al Nat Protoc. 2018). By discovery of RNAs where adenosines have been edited to iosines, the RNAs that were bound by the protein of interest and then edited by ADAR are identified genome-wide. We conducted this analyis for DDX41, an RNA Helicase protein with multiple described cellular functions, including RNA splicing and innate immune sensing. Because DDX41 mutations have been discovered in leukemia patients, this analysis was conducted in the human lyeloid leukemia cell line THP-1.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178892 | GEO | 2022/06/01

REPOSITORIES: GEO

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Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [hyperTRIBE]

Project description:Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence. To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada).

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HyperTRIBE Analysis for DDX41 in human leukemia cells

Project description:HyperTRIBE Analysis for DDX41 in human leukemia cells

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HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [Molm13_ADA_RRM_controls]

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HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [MOLM-13]

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HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [LSC and LSK]

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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq]

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