Fig. 1

FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C^WT (lanes 8–13), but not its catalytic mutant FAM46C^mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using ³²P-labeled (A)₁₅ as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C^WT and its catalytic mutant FAM46C^mut. c FAM46D^WT is an active poly(A) polymerase in vitro and requires Mn²⁺ ions for its activity. Purified protein was incubated with ³²P-labeled (A)₁₅ primer in the presence of ATP and divalent cations as follows: Mg²⁺ (lanes 4–6), both Mg²⁺/Mn²⁺ (lanes 7–9), or Mn²⁺ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked ³²P-labeled RNAs co-purified with FAM46C^WTGFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram