Figure 3.
Methylation of Chtop regulates its interactions with export factors. (A) FLAG-tagged Chtop was expressed in 293T cells grown in the presence or absence of the methylation inhibitor AdOx. At the indicated time points, 10 μg of total cell extracts was analysed by western blot using anti-FLAG antibody. (B) Western blot analysis of 293T cells incubated with 10 μg/ml cycloheximide for 8 h. Whole cell extracts of cells grown ±AdOx were analysed (lanes 1 and 2). Additionally, untreated cells were lysed in a buffer ±AdOx and nuclear and cytoplasmic fractions were analysed with the indicated antibodies (lanes 3–8). Ssrp1 western blotting was used to confirm that cytoplasmic fractions were not contaminated with nuclear material. (C) Gst-Uap56, Gst-Alyref and Gst-Nxf1 were used in pulldown assays with 293T cell extract with/without AdOx in the presence of RNase A. Proteins were detected by western blot using a Chtop monoclonal antibody. (D) IP of Nxf1 in the presence of RNAse A from 293T cells treated with AdOx as indicated. Proteins were detected by western blotting with anti-Chtop and anti-Nxf1. (E) Gst-Uap56, Gst-Alyref and Gst-Nxf1 were used in pulldown assays with recombinant Chtop alone or co-expressed with Prmt1 in the presence of RNase A. Proteins were detected via Coomassie staining and western blot (top panel). (F) Chtop only binds Nxf1 when methylated, even in the presence of Alyref. Gst-pulldown assays were carried out with the indicated fusion proteins using 293T cell lysate treated as indicated with AdOx. Chtop was detected using anti-Chtop monoclonal antibody (top panel). The secondary antibody used for western detection also detects the Gb1-6His-Alyref by virtue of the Gb1 tag. (G) mRNP capture assay. Poly(A)+ RNAs from 293T cells grown ±AdOx were purified on oligo-(dT) beads in denaturing conditions after UV crosslinking (+) or not (−). Total extract (1% of input) and eluted proteins were analysed by western blotting with Chtop antibody.
Source data for this figure is available on the online supplementary information page.
