ZFP36 mRNAs are readenylated through poly(U)-containing 3′-UTR elements upon macrophage activation. (A) Genome browser tracks of TED-seq reads for ZFP36 in the presence or absence of Actinomycin D (ActD) at different time-points post-activation (y-axis), together with 3′-seq at the same time-points. One of two biological replicates is shown on a genome browser for TED-seq and 3′-seq. (B) PAT assay (tail length, y-axis) for ZFP36, during LPS activation time-course in the presence or absence of ActD (one of three replicates shown as a representative). Red asterisk indicates the PCR product for completely deadenylated mRNAs, derived from RNase H treatment in the presence of oligo dT. (C) Schematic of GFP reporters with either wild-type (WT) or mutant versions (MUT-DEL, MUT-GC) of human ZFP36 3′ UTR. Three distinct THP-1 stable cell lines were generated with each expressing one of the GFP-ZFP36 reporters by lentiviral transduction. Known RBP motifs were searched in the 3′-UTR region. For the motifs containing consecutive Us (≥3 Us) and with at least half of the motif length composed of Us (annotated in red), the consecutive Us were modified to have a deletion or GC substitution (annotated in green). The 3′-UTR length is indicated under each construct name in brackets. The PAT assay forward primer is represented as a red line for each construct with their distances from the cleavage site in brackets. (D) PAT assay on the human ZFP36 3′-UTR reporter mRNAs of WT, MUT-DEL, and MUT-GC versions expressed in the differentiated THP-1 cells before and after LPS treatment (1 h). The gel image of PAT assay (left panel) was digitally quantified by image J (right panel). Red asterisk denotes the PCR product of completely deadenylated mRNA (A0). See also Supplemental Figure S7.
