Figure 2: Exportin Nxt1 and Nxf1 are involved in flam RNA export and intra-nuclear traffic to Dot COM.

(a) Quantitative analysis of flam transcript localization in ovarian follicle cells. The percentage of cells with a nuclear (left), transmembrane (middle) and cytoplasmic (right) focus is plotted. Error bars represent s.e.m. n=45 (ISO1A), n=28 (Nxt1), n=22 (Nxf1), n is the number of independent experiments. ****P value <0.0001 and **P value <0.01 are calculated according to Mann–Whitney test. (b) flam transcripts (red) and nuclear membrane (green) are visualized using flam 508 RNA probe and anti-lamin antibody, respectively. One Z-stack of confocal image is shown. Therefore, as flam foci are not all in the same focus they cannot be all visualized in every nucleus of the field. (c) Fold change in steady-state RNA level of flam in Nxt1-SKD ovaries compared with ISO1A ovaries. RNA levels are normalized to rp49 level. The positions of the primers used are indicated above and their sequence is given in Supplementary Table 2. Data are presented as means (n=3). Error bars indicate s.e.m. *P value <0.05, according to the Student’s t-test. (d,e) Changes in steady-state levels of flam unique 23–29 nt piRNAs in ovarian cells of ISO1A and Nxt1- and Nxf1-SKD lines measured by small RNA sequencing (normalized to one million genome-mappable reads). (d) Density plot of unique 23–29 nt piRNAs mapping to the flam piRNA cluster. The Y and X axes in e are identical for the three graphs. X axis represents the flam 180 kb starting from its transcription start site on the left. Y axis represents the quantity of piRNAs normalized to one million genome mappable reads. (f) Quantitative analysis of relative localization between flam DNA and flam transcripts in follicle cells. The percentage of cells with a DNA–RNA co-localization is plotted. Error bars represent s.e.m. n=32 (ISO1A), n=20 (Nxt1), n=38 (Nxf1), n is the number of independent experiments. ****P value<0.0001, ***P value <0.001), according to Mann–Whitney test. Error bars represent s.d. *P value <0.05, according to Mann–Whitney test. (g) Double DNA/RNA FISH experiments. flam DNA (green) and flam transcripts (red) are respectively detected with DNA probe and 508 riboprobe. DNA is stained with Hoescht (white).