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

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).