Figure 3—figure supplement 1. | Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

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Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

Figure 3—figure supplement 1.

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Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
Figure 3—figure supplement 1.
Download figureOpen in new tabFigure 3—figure supplement 1. MEG-3 RNA binding.

(A) Coomassie stained gel showing His-tagged MEG-3 proteins. Expected sizes are indicated on the right. Each lane is from a separate gel. (B) RNA-RNA competition EMSA. 300 nM MEG-3 (300 nM) was incubated with 50 nM poly-U30 and increasing amounts of unlabeled 30-mer RNAs as indicated to the left of each panel. The first lane of each gel contains only labeled poly-U RNA. The last lane of each gel contains poly-U RNA and the highest concentration of unlabeled competitor RNA (5000 nM). The unbound poly-U RNA is denoted by an asterisk (*). Double asterisks (**) in the poly-G assay denote a band that arises due to an interaction between poly-G and poly-U (independent of MEG-3). For each image shown, n = 2 technical replicates. (C) Fluorescence Polarization of polyuridine by MEG-3 (expanded from Figure 3). Fluorescence polarization values normalized relative to saturation are shown for full length MEG-3 (top, green), MEG-3IDR (middle, black), and MEG-3Cterm (bottom, red). Values represent averages of ≥3 technical replicates. A fit of the polarization as a function of protein concentration is plotted and used to calculate the given Kd,app. Error bars report S.E.M.

DOI: http://dx.doi.org/10.7554/eLife.21337.008