Fig 2: Repressing the editing of endogenous sites by SRSF9. (A) Schematic of our experimental setup to evaluate whether SRSF9 could repress ADAR1-mediated editing and ADAR2-mediated editing. (B) Effect of SRSF9 on A-to-I editing of endogenous sites in HEK293T cells. Hg19 coordinates are shown. Genes highlighted in red are not brain-specific. For SON, chr21:34922806 and chr21:34923225 were not edited in HEK293T cells (even with ADAR2 overexpression). Hence, we focused mainly on chr21:34922801 and chr21:34923319. Additionally, we discovered novel editing sites (indicated by asterisks next to their coordinates) in GRM4, KCNA1 and NOVA1. Error bars indicate the s.e.m. (n ≥ 3 biological replicates). (*P < 0.1, **P < 0.05, ***P < 0.01; Student’s t-test. All significant sites must show at least 5% change in their editing levels). (C) Chromatograms showing SRSF9 inhibiting the editing of protein-coding sites in a human neural progenitor cell line ReN-VM. The editing sites, chr21:34922801 (SON), chr6:44120349 (TMEM63B) and chr3:58141801 (FLNB) were not edited in ReN-VM.

Fig 3: Suppression of ADAR2-mediated editing of CaV1.3 by SRSF9. (A) Top, schematic diagram of gIQECS minigene reproduced from Figure 1B. Bottom, bar chart reporting ADAR2-mediated editing of the transcripts expressed by full-length gIQECS minigene upon co-expression with RPS14, DHX15, and SRSF9 in heterologous HEK293 expression system. Editing of ATA and TAC codons were normalized against ADAR2 transfection alone, before averaging. Values shown are mean ± S.E. (n = 3). (B) Normalized editing level upon co-expression of increasing amount of SRSF9 with the same amount of ADAR2, format as in (A). (C) Normalized editing level upon co-transfection of 1, 2 and 4 μg of ADAR2 in the presence of 2 μg of SRSF9, format as in (A). (D) Over-expression of ADAR2 in neuroblastoma SH-SY5Y cells significantly increased the editing level of endogenous human CaV1.3 IQ-domain which could be suppressed with co-expression of SRSF9. Bar chart reporting the percent editing at ATA and TAC codon of human CaV1.3 under respective conditions. ***P < 0.001 and ns, non-significant as compared to the vector control (Student's unpaired t-test) Values shown are mean ± S.E. (n = 3).

Fig 4: Knockdown of ADAR2 increases rectification of AMPAR currents.(A) Representative traces from an RGC transfected with ADAR2 SiRNA (Ai) and another transfected with scrambled SiRNA (Aii). Both cells were recorded from with a pipet solution containing 300 µM spermine, which blocks CP-AMPAR currents at positive, but not negative voltages. Responses to 50 ms puffs of AMPA (arrow) at −60 mV have been adjusted to approximately equal size so that the larger response to AMPA at 40 mV in the RGC transfected with scrambled SiRNA is apparent. (B) Summary I–V relation for ADAR2 (filled symbols, n = 14 cells) and scrambled SiRNA (open symbols, n = 12 cells). Responses in both groups have been normalized to the responses at −60 mV for each cell. Continuous line is fitted to the mean amplitude of the AMPA current at −60 and −10 mV for cells transfected with scrambled SiRNA. (C) Data from (C) replotted as the rectification index (RI), calculated as described in the Methods. The smaller number indicates a greater degree of rectification for RGCs transfected with ADAR2 SiRNA as compared to RGCs transfected with control, scrambled SiRNA (*indicates p<0.01). The data are consistent with the idea that knockdown of ADAR2 with SiRNA increases expression of CP-AMPARs in RGCs.

Fig 5: Role of ADAR1 and ADAR2 in Vasoactive MicroRNA Editing and Maturation(A–E) ADAR1 and ADAR2 were knocked down in HUAFs through transfection of an ADAR1-targeting or ADAR2-targeting siRNA (siADAR1 and siADAR2, respectively). The subsequent effects on microRNA expression and editing were analyzed as in Figures 2 and 3 and compared to transfection with a negative control siRNA (siNegCtrl). (A) pri-MicroRNA A-to-I editing and expression. (B–E) Mature (B) miR-376a&b-3p, (C) miR-376c-3p, (D) miR-381-3p, and (E) miR-411-5p expression relative to U6 (top panels) and the corresponding percentage of mature microRNA-ED (bottom panels). All data are presented as mean ± SEM from three independent experiments performed with pooled cells from 13 different umbilical cords. #p < 0.01, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, versus siNegCtrl or as indicated by a two-sided Student’s t test.