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. 2021 Mar 2;10:e64252. doi: 10.7554/eLife.64252

Figure 1. RNA determines the morphology of dynamic mesh-like RNA granules in cells.

(A) Confocal live-cell imaging of HeLa cells after the transfection of mCherry-tagged TIS11B. GFP-SEC61B was co-transfected to visualize the endoplasmic reticulum. The white dotted line demarcates the nucleus. Right: higher magnification of the indicated region. Scale bars, 5 µm (overview) and 1 µm (zoom-in). (B) Same as (A), but after transfection of TIS11B with a mutated RNA-binding domain. See Figure 1—figure supplement 1 for more mutants. CC: C135H/C173H. (C) Same as (A), but after transfection of mCherry-tagged SUMO10-SIM5 or SUMO-SIM-TIS chimera. 73% (N = 52) of SUMO-SIM-TIS granules are mesh-like. (D) Same as (A), but after transfection of mGFP-tagged FUS IDR (amino acids 1–214) or FUS-TIS chimera. All granules are mesh-like. (E) Fluorescence recovery after photobleaching of FUS-TIS and SUMO-SIM-TIS 16 hr after transfection of mCherry-SUMO-SIM-TIS or mGFP-FUS-TIS into HeLa cells. Scale bar, 1 µm.

Figure 1.

Figure 1—figure supplement 1. Mutation of the TIS11B RNA-binding domain generates sphere-like granules in cells.

Figure 1—figure supplement 1.

(A) Fluorescence recovery after photobleaching of TIS11B 5 hr after transfection of mGFP-TIS11B into HeLa cells. Scale bar, 1 µm. (B) Shown is the amino acid sequence of the wild-type (WT) TIS11B RNA-binding domain (RBD), together with the introduced point mutations (red). CC: C135H/C173H; FF: F137N/F175N; KK: K116L/K154L; RK: R114L/K152L. (C) Western blot showing RNA oligonucleotide pulldown after co-transfection of the indicated mCherry-tagged TIS11B constructs and an AU-rich RNA oligo (TNFα AU-rich element) into HeLa cells. RNA-binding is disrupted in CC, FF, and KK mutants. HuR also binds to the AU-rich element. Tubulin was used as loading control. 2.5% of the input was loaded. (D) Confocal live-cell imaging of HeLa cells after transfection of the indicated constructs described in (B). GFP-SEC61B was co-transfected to visualize the endoplasmic reticulum. Scale bar, 1 µm. (E) Quantification of the data shown in (D). Shown is the fraction of cells with sphere-like granules formed by the indicated TIS11B proteins.
Figure 1—figure supplement 2. In the context of various multivalent domains, the TIS11B RNA-binding domain generates mesh-like condensates in vivo.

Figure 1—figure supplement 2.

(A) Confocal live-cell imaging of HeLa cells after the transfection of mCherry-tagged TIS11B RNA-binding domain (RBD). GFP-SEC61B was co-transfected to visualize the endoplasmic reticulum (ER). The white dotted line demarcates the nucleus. Right: higher magnification of the indicated region. Scale bars, 5 µm (overview) and 1 µm (zoom-in). (B) Confocal live-cell imaging of HeLa cells after transfection of mCherry-tagged-SUMO-SIM-TIS. GFP-SEC61B was co-transfected to visualize the ER. Scale bar, 1 µm. (C) Representative images obtained from RNA-FISH (green) against GFP after transfection of GFP-CD47-LU or GFP-CD274-3′UTR in HeLa cells. BFP-FUS-TIS (magenta) was co-transfected. The white dotted lines demarcate the cell border and the nucleus, respectively. Right: line profiles of fluorescence intensities including the Pearson’s correlation coefficient (r). The arrow indicates the plane used for line profile generation. Scale bar, 5 µm.