Figure 4. RNA polymerase I as a novel survival motor neuron (SMN)–interacting protein.

(A) SMN protein interacts with RNA polymerase I. Immunoprecipitation (IP) experiments were carried out in HEK293T cell nuclear extract using anti-RNA polymerase II, anti-RNA polymerase I, or control rabbit (IgG) antibodies. Immunoprecipitates were analysed by SDS–PAGE and Western blotting with antibodies against RNA polymerase II, RNA polymerase I, SMN, and senataxin. (B) SMN–RNA polymerase I interaction is RNA independent. Immunoblot analyses of RNA polymerase I, senataxin, SSRP1, and SMN on immunoprecipitations with RNA polymerase I (RNA polymerase I IP) from nuclear extracts of HEK293T cells without and with RNAse A treatment, respectively. Rabbit IgG was used as negative control. (C) In situ proximity ligation assay detection of the interaction between SMN and RNA polymerase I in HEK293T cells. Scale bars represent 5 μm. Quantification of the number of puncta per nucleus was performed. Bar graphs of mean ± SEM (N = 3). Kruskal–Wallis non-parametric test with Dunn’s multiple comparisons. ***P ≤ 0.001, and ****P ≤ 0.0001. (D) Schematic representation of EGFP-tagged SMN construct used in the experiments. (E) IPs were performed with nuclear extracts from HEK293T cells transiently transfected with EGFP-tagged SMN full length, SMN E134K, SMN Δ3, and SMN Δ7, respectively. The anti-GFP incubation served as a loading control.

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