Fig. 3

FAM46C tethering leads to polyadenylation and enhanced expression of a Renilla luciferase (RL) reporter. Analysis at the protein level (a–d): a FAM46C tethering increases RL reporter protein levels. HEK293 cells were co-transfected with a construct expressing RL, containing five boxB sites in its 3′-UTR, Firefly luciferase (FL) control reporter and FAM46C^WT harboring the N-terminal λN boxB-binding domain and HA-tag. Western blot detection of RL in mock-transfected cells (lane 1) and after NHA-FAM46C^WT (lane 2) or NHA-FAM46C^mut (lane 3) tethering. Expression of NHA-tagged FAM46C proteins were confirmed using an α-HA antibody. DBC1 served as a loading control. Asterisks indicate cross-hybridization signals. b Expression of RL5boxHSL + HhR reporter with a cyclic phosphate at the 3′ end generated by a hammerhead ribozyme was not enhanced upon FAM46C tethering. The experiment was performed as in a. c Expression of the FAM46C without λN domains did not enhance expression of the reporter. The experiment was performed as in a. d Quantifications of RL protein normalized by the internal FL reporter related to experiments from a–c. RNA analysis (e–i): e northern blot detection of RL mRNA using total RNA from HEK293 cells after tethering of NHA-FAM46C^WT or NHA-FAM46C^mut to RL5box (lanes 1–3) or RL5boxHSL + HhR with a cyclic phosphate at the 3′ end (lanes 4–6). f Quantifications of RL mRNA. g RT-qPCR analysis of the FL control reporter. h Northern blot detection of Renilla luciferase using poly(A) + fraction from HEK293 cells after tethering of NHA-FAM46C^WT or NHA-FAM46C^mut (i) Poly(A) tails added to reporter mRNA can be removed by RNase H treatment in the presence of oligo(dT)₂₅. High-resolution northern blot analysis of RL mRNA from control HEK293 cells (lanes 1–2), after tethering of NHA-FAM46C^WT (lanes 3–4) or NHA-FAM46C^mut (lanes 5–6). The data in d, f, g are shown as a mean value ± SD (n = 3)