Fig. 2: slncRNAs and proteins can form RNA-protein granules in vitro.

a Construct diagram depicting the suspension of tdPCP-mCherry recombinant protein together with in vitro transcribed slncRNA, resulting in synthetic RNA-protein granules. b Microscopy images showing an overlay of the 585 nm channel (mCherry) and the 488 nm channel (slncRNA). All scale bars are 10 µm. c Boxplots of median 585 nm (mCherry) fluorescence intensity values collected from multiple granules. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The value for ‘Whisker’ corresponds to ±1.5 IQR (interquartile rate) and extends to the adjacent value, which is the most extreme data value that is not an outlier. The outliers are plotted individually as plus signs. d Top - median 488 nm (Atto488) fluorescence intensity values collected from multiple slncRNA granules (blue) and slncRNA-protein granules (orange). Quenching for the slncRNA granules is empirically estimated at 1x for the PCP-4x and PCP-3x/MCP-3x, 1.34x for the PCP-4x/MCP-4x, 1.37 for the PCP-8x, and 4.2x for PCP-14x/MCP-15x. Note that we assume no quenching for the SRNP granules, except for the case of PCP-14x/MCP-15x. Data presented as median values ± SEM. Bottom—Increase in Atto-488 fluorescence between slncRNA-protein granules and slncRNA only granules, for the different slncRNA molecules. Data in top and bottom panels was collected from: 112 PCP-3x/MCP-3x, 165 PCP-4x, 204 PCP-4x/MCP-4x, 121 PCP-8x, and 89 PCP-14x/MCP-15x, RNA-only granule, and from 91 PCP-3x, 69 PCP-3x/MCP-3x, 30 PCP-4x, 92 PCP-4x/MCP-4x, 85 PCP-8x, and 37 PCP-14x/MCP-15x RNA-protein granules. e Structured illumination super resolution images of (left) PCP-14x\MCP-15x slncRNA-protein granule, and (right) PCP-4x slncRNA-protein granules. Color bar indicates fluorescence intensity. f Structured illumination super resolution images of PCP-14x/MCP-14x slncRNA-only granules. Scale bar is 2 µm. Color bar indicates fluorescence intensity. g Microscopy images for serial dilutions of reaction components taken at T = 1 hr after reaction setup. Highest concentrations show the formation of highly fluorescent filamentous structures, as seen in the top left image. Lower RNA concentrations result in smaller structures, while lower protein concentration result in weaker fluorescence. Scale bar is 10 µm. Due to high dynamic range, the intensities presented are the square root of the raw data images. h Maximal observed intensity values for each reaction condition at time T=0 and T=1 hr. All distributions were derived from 5 separate microscopy images of granule reaction prepared with the listed concentrations. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The value for ‘Whisker’ corresponds to ±1.5 IQR (interquartile rate) and extends to the adjacent value, which is the most extreme data value that is not an outlier. The outliers are plotted individually as plus signs. Source data are provided as a Source data file.