Fig. 3: A 29 bp 3’UTR motif directs cell-size-scaling of TRAK2 mRNA distribution.

a Sequences of TRAK2 3’UTRs from Wt and ΔTRAK2 ECs indicating the location of gRNA target (red), NGG PAM (blue), and G-motif sequences (magenta). b PCR of the TRAK2 3’UTR demonstrating a band shift in ΔTRAK2 ECs (n = 3 experimental repeats). c, d smFISH co-detection of TRAK2 and GAPDH mRNAs in Wt (c) or ΔTRAK2 (d) ECs (red circles indicate mRNA spots; arrowheads indicate distal mRNA accumulation; brackets indicate distal-most 10 µm, 20 µm, and 30 µm of protrusions; dashed line indicates cell outline). e Number of TRAK2 mRNA spots in Wt and ΔTRAK2 ECs (n = 31 cells Wt, n = 28 cells ΔTRAK2, two-tailed Mann–Whitney test, ns P = 0.1130). f, g Relative distribution of TRAK2 mRNAs (f) or GAPDH mRNAs (g) in Wt and ΔTRAK2 EC protrusions (n = 31 cells Wt TRAK2, n = 28 cells ΔTRAK2 TRAK2, n = 26 cells Wt GAPDH, n = 25 cells ΔTRAK2 GAPDH, two-tailed Mann–Whitney test, ***P ≤ 0.0001, **P ≤ 0.0096 for 10–20 µm in f, 0.0044 for 0–10 µm in f, ns P = 0.2848 in f, 0.591 for proximal in g, 0.556 for 20–30 µm in g, 0.3945 for 10–20 µm in g, 0.8702 for 0–10 µm in g). h Distance the RNA CoM sits from the nucleus for GAPDH and TRAK2 mRNAs in Wt and ΔTRAK2 ECs (n = 29 cells Wt GAPDH, 26 cells ΔTRAK2 GAPDH, n = 32 cells Wt TRAK2, 37 cells ΔTRAK2 TRAK2, two-tailed Mann–Whitney test, ***P ≤ 0.0001, ns P = 0.2793). i Protrusion length in Wt and ΔTRAK2 ECs (n = 31 cells Wt, n = 28 cells ΔTRAK2, two-tailed Mann–Whitney test, ns P = 0.3282). j Distance the RNA CoM sits from the nucleus normalised to protrusion length for GAPDH and TRAK2 mRNAs in Wt and ΔTRAK2 ECs (n = 33 cells Wt GAPDH, 30 cells ΔTRAK2 GAPDH, n = 32 cells Wt TRAK2, 37 cells ΔTRAK2 TRAK2, two-tailed unpaired t test, ***P ≤ 0.0001, ns P = 0.8949). k Plots comparing the distance that the RNA CoM sits from the EC nucleus versus protrusion length for GAPDH and TRAK2 mRNAs in either Wt or ΔTRAK2 ECs (n = at least 26 cells, two-sided analysis of covariance, ***P ≤ 0.0001 versus GAPDH, ns P = 0.3410 versus GAPDH). l Plot comparing the distance that the TRAK2 RNA CoM sits from the nucleus in Wt and ΔTRAK2 ECs with protrusion length (n = 32 cells Wt, n = 37 cells ΔTRAK2, two-tailed unpaired t test, ***P = 0.0002 for 50–70 µm, <0.0001 for 60–80 µm, **P = 0.0067, ns P = 0.1523 for 20–40 µm, 0.0753 for 30–40 µm, 0.0530 for 40–60 µm Wt versus ΔTRAK2; two-sided analysis of covariance, magenta asterisk, ***P = 0.0005 Wt versus ΔTRAK2). All cells cultured on CDM except k, l where data for cells cultured on glass and CDM were pooled. Data are mean ± s.e.m. (e–j, l). c, d Scale bar, 10 µm. Source data are provided as a Source Data file.