Fig. 1. TENT-5 is a noncanonical cytoplasmic poly(A) polymerase in worms.

(A) Phylogenetic relationship among 48 TENTs from five model organisms (Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis; Dm, Drosophila melanogaster; Ce, C. elegans). (B) Schematic illustration of RNA tethering assay, modified from (27). A protein of interest (TENT-5^{WT/D151A,D153A}) binds to a RL reporter mRNA through an artificial protein-RNA interaction (tether). For tethering assays described in this work, 293T cells were cotransfected with a construct that expresses RL, containing a tether binding site (five boxB sites) in its 3′UTR, and a construct expressing either wild-type or catalytically inactive TENT-5 harboring the N-terminal λN boxB-binding domain and an HA-tag (NHA). (C and D) DRS-based poly(A) length profiling of mRNA following tethering assay. Tethering of wild-type TENT-5 led to increased polyadenylation of an RL reporter mRNA compared to a tethering with a catalytically inactive TENT-5^D151A,D153A mutant. Shown are density distribution plots for RL mRNA (C) and all other transcripts detected in 293T cells (D) scaled to 1. Vertical dashed lines represent median poly(A) lengths (in nucleotides). (E and F) Representative fluorescence and differential interference contrast (DIC) microscopy images of TENT-5–GFP expression in embryos and adult tent-5::gfp knockin worms (tent-5(rtt6[tent-5::gfp::3xflag] I). ph, pharynx; in, intestine; rec, rectum. Scale bars, 20 μm.