Fig. 2: The foldedness of nos 3′UTR is similar inside and outside of germ granules.

a Predicted secondary structure of nos TCE outside the germ granules with a mapped normalized DMS reactivity generated by DMS-MaPseq. The normalized DMS reactivities were based on an average of two biological replicates (see Supplementary Fig. S1h, i for the correlation coefficients between the two replicates). b An example of the predicted secondary structure of nos 3′UTR outside the germ granules with mapped reactivity by DMS-MaPseq (an average of two replicates is shown). Blue dashed box: nos TCE. c Correlation of the normalized DMS reactivities between inside (y-axis) and outside of germ granules (x-axis). Each purple dot represents the DMS reactivity of the same probed base on nos 3′UTR recorded inside (y-axis) and outside of germ granules (x-axis). d The ratio of the normalized DMS reactivity between inside and outside of the germ granules for the same probed bases on nos 3′UTR. Black dotted line: no change in DMS reactivity. Blue dots and lines: ratio of DMS reactivities between inside and outside of germ granules for the same probed bases. Orange dashed lines represent thresholds with a p-value < 5.68 × 10⁻⁵ (adjusted for multiple hypothesis testing; two-tailed; see “Methods”), at which the nucleotides are significantly more exposed inside (above 1) or outside the granules (below 1). Gray blocks: no data due to gaps in PCR amplification (base positions: 595–657) or primer attachment (base positions: 1–22 and 847–880). The bases, which were significantly more exposed inside the granules, were labeled. e The genotypes of the female flies that laid the eggs for the experiments shown in eii (i). Schematic of a nos mRNA fused with a nos localization element + 2 and an antisense nos 3′UTR and a chimeric mRNA containing a CDS of IRFP fused with WT nos 3′UTR. The CDS of both mRNAs were hybridized with spectrally distinct smFISH probes (green for nos ORF and magenta for IRFP ORF) (ii). The two mRNAs display low co-localization in germ granules (iii), with a PCC(Costes) of 0.32 ± 0.01 (iv). n = 14 embryos. Data: Mean ± SEM. f Endogenous nos hybridized with spectrally-distinct smFISH probes (green and magenta), which alternately hybridized with the nos ORF(i), displaying high co-localization (ii), with a PCC(Costes) of 0.93 ± 0.01 (iii). n = 7 embryos. Data: Mean ± SEM. Scale bars: in all images 2 μm. This experiment served as a positive control to establish the PCC(Costes) threshold for high colocalization. It was conducted in a wild-type (WT) background. See also Supplementary Figs. S1–S3 and Supplementary Data S2 and S3. Source data are provided as a Source Data file.