Multivalent Interactions with CCR4-NOT and PABPC1 Determine mRNA Repression Efficiency by Tristetraprolin

We observe interaction between the RNA-binding protein tristetraprolin (TTP) and the C-terminal end of the NOT1 subunit of the CCR4-NOT complex in a relocalization assay in HEK293-T cells. Here, we immobilize mCherry-tagged TTP at the plasma membrane (PH-tag) and observe relocalization of mEGFP-tagged NOT1-SHD (NOT1, residues 1833-2361) to the plasma membrane. This interaction is resistant to competition with a short fragment of Nanos3 (residue 1-117) previously shown to stably interact with the NOT1-SHD, as seen when ER-localized Nanos3 was co-expressed with PH-tagged TTP and NOT1-SHD.Live-cell imaging was conducted ca. 24 h post-transfection using a 63X, oil, 1.4 NA, 0.19 mm objective on a Zeiss LSM 780 confocal microscope (resolution 1024x1024) in a 37 °C incubation chamber. ZEN 2012 SP5 (version 14.0.20.201) was used for image acquisition.