Fig. 2: RNAs co-localize with Z-disks and microtubules.

A Representative image of IF/FISH co-labeling of Ttn RNAs (red), tubulin protein (microtubules, green), and telethonin protein (Z-disks, blue) in an isolated myofiber. Scale bar: 5 µm. B Zoomed-in regions of IF/FISH co-labeled myofibers as in A for each RNA studied. Scale bars: 1 µm. C Schematic describing the computational pipeline used to assess mRNA proximity to cytoskeleton from images of IF/FISH co-labeled myofibers (A). D Distances from cytoplasmic spots (blue boxes) to Z-disks (left) and microtubules (right) compared to null distributions generated from randomly selected cytoplasmic coordinates (gray boxes). Data from n = 10 myofibers (Polr2a, Hist1h1c, Ttn, GFP) or n = 9 myofibers (Vcl, Dmd, Hnrnpa2b1, Myom1, Gapdh). Spots located within 2 pixels (~0.1 μm) of either filament were considered “cytoskeleton co-localized” (purple shaded region, percentages). Box plots show minimum, first quartile, median, third quartile, and maximum. *p < 0.03, **p < 10⁻⁴, Two-sided Mann–Whitney U test. E Distances from cytoskeleton-associated spots to ZMIs (blue boxes) compared to a null distribution generated from randomly selected coordinates along the cytoskeleton (gray boxes). Number of myofibers same as D. Box plots show minimum, first quartile, median, third quartile, and maximum. *p < 0.03, **p < 10⁻⁴, Two-sided Mann–Whitney U test.