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ZFP36L2 orchestrates stress-adaptive plasticity in intestinal regeneration and colorectal cancer metastasis (HyperTRIBE)

ABSTRACT: HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) was used to identify genome-wide mRNA targets of ZFP36L2 in patient-derived colorectal cancer (CRC) and normal colon organoids. Full-length wild-type ZFP36L2 or the RNA-binding-incompetent frameshift mutant fsZFP36L2 (G144Afs*43) were fused to the hyperactive E488Q mutant catalytic domain of adenosine deaminase acting on RNA (ADAR) and expressed under a doxycycline-inducible Tet-on promoter, with a P2A-EGFP reporter to mark expressing cells. Constructs were stably integrated by lentiviral transduction into four organoid lines: two CRC liver metastasis lines (MSK107Li, OKG146Li), one matched primary tumour line (OKG146P), and one matched normal colon line (OKG146N). On day 5 post-seeding, organoids were treated with 2 μg/mL doxycycline for 2 days to induce transgene expression. GFP-positive live cells were isolated by flow cytometry, and total RNA was extracted using the RNeasy Mini Kit (Qiagen). mRNA libraries were prepared using the TruSeq Stranded mRNA Kit and sequenced on the Illumina NovaSeq 6000 (PE100). ZFP36L2-bound mRNAs were identified by comparing A-to-I editing events (read as A-to-G substitutions) enriched in ZFP36L2-ADAR versus fsZFP36L2-ADAR expressing organoids. Over 80% of ZFP36L2-specific edits occurred in mRNA 3′UTRs, predominantly at the conserved UAUUUA motif, consistent with known ZFP36 family RNA-binding properties.

ORGANISM(S): Homo sapiens

PROVIDER: GSE336416 | GEO | 2026/07/20

REPOSITORIES: GEO

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ZFP36L2 orchestrates stress-adaptive plasticity in intestinal regeneration and colorectal cancer metastasis (ActDseq)

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2026-07-20 | GSE336415 | GEO

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ZFP36L2 orchestrates stress-adaptive plasticity in intestinal regeneration and colorectal cancer metastasis (PDO scRNAseq)

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2013-06-15 | E-GEOD-47945 | biostudies-arrayexpress

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ZFP36L2 orchestrates stress-adaptive plasticity in intestinal regeneration and colorectal cancer metastasis

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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).

2023-07-26 | GSE224485 | GEO

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