Fig. 4: Exposed GC-rich complementary sequences enhance interactions among engineered nos mRNAs in vivo.

a Schematics of the endogenous nos containing four stems with non-palindromic (nos-non), HIV- (nos-HIV) or nos-derived (nos-nos) palindromes in its 3′UTR (demarcated with an orange arrow). b Schematics of unspliced or spliced nos coding sequence (CDS) where the primer targeted regions were shown in magenta arrows (i). Schematic of the crossing scheme with the nos-non experiment shown as an example (ii). nos PCR products from the cDNAs (100 ng total input) of the embryos laid by WT/nos^BN (top), nos-non/nos^BN, nos-HIV/nos^BN and nos-nos/nos^BN flies (bottom). Three lanes per each genotype represent three biological replicates (iii). c Schematic of the primer targeting regions (1, 2, 3 and 4) on nos-non, which are also used for WT nos, nos-HIV and nos-nos (iii). The expression levels of each region are determined by qRT-PCR (ii). For biological replicates, n = 6 and 3 embryo samples for regions 1 and 3, 2 and 4, respectively. Data: Mean ± SEM. n.s.: not statistically significant. p-values: 0.0022 (region 1: nos-non/nos^BN vs. nos-HIV/nos^BN; Mann-Whitney; two-tailed; **), 0.94 (region 1: nos-non/nos^BN vs. nos-nos/nos^BN; Mann-Whitney; two-tailed), 0.10 (region 2: nos-non/nos^BN vs. nos-HIV/nos^BN; Mann-Whitney; two-tailed), 0.70 (region 2: nos-non/nos^BN vs. nos-nos/nos^BN; Mann-Whitney; two-tailed), 0.080 (region 3: nos-non/nos^BN, nos-HIV/nos^BN and nos-nos/nos^BN; Kruskal-Wallis) and 0.30 (region 4: nos-non/nos^BN, nos-HIV/nos^BN and nos-nos/nos^BN; Kruskal-Wallis). d Schematic of the Drosophila embryo and mRNA clusters in its germ granules. Created in BioRender. Trcek, T. (2025) https://BioRender.com/xdghx4d. e smFISH of nos-non, nos-HIV, and nos-nos mRNAs in soma of the embryos laid by nos-non/nos^BN, nos-HIV/nos^BN and nos-nos/nos^BN females (top). Heat maps were generated based on the number of mRNAs per cluster (bottom). Scale bars: 5 μm. f Concentrations of nos-non (n = 7 embryos), nos-HIV (n = 6 embryos) and nos-nos (n = 6 embryos) mRNAs in soma. Data: Mean ± SEM. n.s.: not statistically significant. p-value: 0.24 (nos-non/nos^BN, nos-HIV/nos^BN and nos-nos/nos^BN; Kruskal-Wallis). g Number of nos mRNAs per cluster in soma of the embryos laid by nos-non/nos^BN (n = 7 embryos), nos-HIV/nos^BN (n = 6 embryos) and nos-nos/nos^BN (n = 6 embryos) flies. Data: Mean ± SEM. h smFISH of nos-non, nos-HIV, and nos-nos mRNAs in the germ plasm of the embryos laid by nos-non/nos^BN, nos-HIV/nos^BN and nos-nos/nos^BN females (top). Heat maps were generated based on the number of mRNAs per cluster (bottom). Scale bars: 20 μm. i Concentrations of nos-non (n = 6 embryos), nos-HIV (n = 6 embryos) and nos-nos (n = 6 embryos) mRNAs in the germ plasm. Data: Mean ± SEM. n.s.: not statistically significant. p-values: 0.026 (nos-non/nos^BN vs. nos-HIV/nos^BN; Mann-Whitney; two-tailed; *) and 0.0022 (nos-non/nos^BN vs. nos-nos/nos^BN; Mann-Whitney; two-tailed; **). j Number of nos mRNAs per cluster in the germ plasm of the embryos laid by nos-non/nos^BN (n = 6 embryos), nos-HIV/nos^BN (n = 6 embryos) and nos-nos/nos^BN (n = 6 embryos) flies. Data: Mean ± SEM. See also Supplementary Fig. S7 and Supplementary Data S4. Source data are provided as a Source Data file.