Fig 2: XRN2 accumulation does not change in excitatory neurons after SE induction.In order to examine whether SE induction changes the accumulation of XRN2 (green) in specific neuronal subpopulations, we conducted double-labeling experiments using anti-CamKIIα (red) in coronal sections of control and SE animals counter-stained with DAPI (blue). (A) In the representative image of the DG it is possible to observe that XRN2 is present in CamKIIα-positive granule cells of the control group. The pixel intensity profile reveals that the green signal is surrounded by the red signal, demonstrating the presence of XRN2 in excitatory neurons of the control group. (B–D) In high magnification of selected area, we confirmed the presence of XRN2 in CamKIIα- positive cells (white arrows) of the DG. (E) We performed the same analysis in the SE group. The pixel intensity profile revealed the same pattern observed in the control group. (F–H) In high magnification of selected area it is possible to observe that the amount of XRN2 in CamKIIα-positive neurons (white arrowheads) is similar to what we observed in the control group. (I–K) Quantification of the mean pixel intensity of XRN2 in CamKIIα-positive neurons of the control group did not show modifications after SE induction in CA1, CA3 and dentate granule cells, respectively. Bars represent standard errors of mean. Scale bar: 25 μm.

Fig 3: DDX5 physically associates with the 5′–3′ exonuclease XRN2, functioning together to repress R‐loops HEK293 cells were transfected with empty pcDNA3 vector (−) or Flag‐DDX5 (+). Whole cell extracts (WCEs) and anti‐Flag immunoprecipitations (IPs) were immunoblotted with anti‐Flag and anti‐XRN2 antibodies.Untransfected U2OS cells were lysed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti‐XRN2 antibodies. The bound proteins were separated by SDS–PAGE followed by Western blotting with anti‐DDX5 or anti‐XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag‐DDX5 WT (1–614) or truncated Flag‐DDX5 proteins (60–614; 1–554; 1–435). WCEs of the transfected cells (left panel) were Western‐blotted with anti‐Flag and anti‐XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed, and anti‐Flag immunoprecipitations (right panel) performed in the presence of co‐immunoprecipitating endogenous XRN2 by Western blotting with anti‐XRN2 antibodies. M denotes molecular mass markers in kDa.Untransfected and U2OS cells stably expressing Flag‐DDX5 WT or Flag‐DDX5‐RK were subjected to immunoprecipitation with anti‐Flag antibody. The WCEs and the anti‐Flag immunoprecipitated proteins were Western‐blotted with anti‐monomethylarginine (MeR), anti‐Flag, and anti‐XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP‐qPCR analysis with anti‐IgG and anti‐S9.6 antibodies with or without RNase H treatment. The average and SEM from three independent experiments are shown. Statistical significance was assessed using Student's t‐test. *P < 0.05; **P < 0.01. Source data are available online for this figure.

Fig 4: XRN2 and PAPD4 accumulate in α-ganglion cells in the ganglion cell layer.(A) To investigate the presence of XRN2 (green) in specific neurons in ganglion cell layer (GCL), we performed double-labeling experiments using anti-parvalbumin (red), a marker for α-ganglion cells located in GCL, in vertical sections of rat adult retinas counter-stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). (B–D) In high magnification of selected areas, we were able to detect the presence of XRN2 in parvalbumin-positive cells (white arrow), and in other cells located in the GCL, as well (white arrowhead). (E) In addition, we also examined the presence of PAPD4 in these cells. (F–H) Likewise, in high magnification of selected areas, we observed the presence of PAPD4 in parvalbumin-positive cells (red arrow), as well as in other cells located nearby in the GCL (red arrowhead). Scale bar: 60 µm.

Fig 5: PAPD4, but not XRN2, is regulated by ambient light levels.(A–C) Using quantitative real time PCR, we compared XRN2 and PAPD4 gene expression levels after 3 and 24 hours of dark-adaptation (3h DA and 24h DA, respectively) and 24 hours of dark-adaptation followed by return to 12∶12 light/dark cycle (DA/RC) for 24 hours (n = 6). We were not able to detect changes in XRN2 and PAPD4 gene expression levels in any experimental condition. In these experiments, GAPDH abundance was used as internal control. (D–F) Interestingly, we observed upregulation in PAPD4 protein levels after 3 hours (160%, P<0.05) and 24 hours (244%, P<0.01) of dark-adaptation when compared to controls. No significant changes were observed in XRN2 protein levels. In these experiments, beta-actin abundance was used as internal control (n = 4). *P<0.05, **P<0.01 vs. control in Tukey's pairwise comparisons after one-way ANOVA.