Figure 1: Role of the 3′-UTR region in FUS regulation.
From: An ALS-associated mutation in the FUS 3′-UTR disrupts a microRNA–FUS regulatory circuitry
(a) Upper panel: construct for Dox-inducible expression of a RFP–FUS fusion protein, stably integrated in SK-N-BE cells16. Lower panel: western blot analysis with FUS antibodies on total proteins extracted from SK-N-BE cells, carrying a RFP control construct (Ctrl) or the RFP–FUS expression cassette, grown for 48 h in absence (−) or presence (+) of doxycycline (Dox). GAPDH was used as a loading control. See full blots with marker position in Supplementary Fig. 2a. (b) FUS cDNA constructs utilized; FUS–WT, contains the wild-type 3′-UTR, while the mutant derivatives contain either the G48A substitution (FUS–G48A) or the deletion of the entire 3′-UTR (FUS-Δ3′-UTR). Histograms show the levels of total FUS protein (left panel) and mRNA (right panel) obtained from SK-N-BE cells transfected with the different FUS constructs. FUS mRNA levels were normalized on the neomycin marker co-expressed from the same plasmid. A representative western blot analysis is shown below the corresponding graph. See full blots with marker position in Supplementary Fig. 2b. (c) Left panel: luciferase fusion constructs. The MRE sequence for mir-141/200a is indicated together with the G48A mutation. Right panel: relative luciferase levels of Luc–FUS–WT, Luc–FUS-Δseed and Luc–FUS–G48A constructs co-transfected in SK-N-BE cells with an empty vector (Ctrl) or with miRNA (miR-141 or miR-200a)-expressing plasmids. (d) Schematic representation of the seed sequence of miR-141 and miR-200a (miR-141/200a WT) and of their mutant derivatives (miR-141/200a mut) containing the complementary substitution to the G48A mutation. Histograms indicate the relative luciferase activity of the Luc–FUS–G48A construct co-transfected in SK-N-BE cells with wild-type (black bars) or mutant (white bars) miRNA-expressing plasmids. Luciferase activity was measured as the ratio between Firefly and Renilla Luciferase activities and shown with respect to a control sample set to a value of 1. In the figure, data were derived from three independent experiments; error bars represent s.e., *P<0.05 corresponds to the one-tailed Student’s t-test, **P<0.01.
