Fig 2: Impact of DENV on RBM10 and its consequences on splicing, inflammation and viral replication. Nuclear RBM10 regulates alternative splicing favoring anti-viral mRNA isoforms, among which SAT1 isoforms were investigated in this report. Upon DENV infection, NS5 intrudes into the spliceosome, as previously reported by our laboratories, and also targets RBM10 for proteasomal degradation, favoring pro-viral splicing isoforms. On the other hand, RMB10 also interacts with viral genome and RIG-I and promotes the ubiquitination of the latter, which is known to be required for its activation and the induction of the innate immune response. Overall, RBM10 is required for the induction of interferon and pro-inflammatory cytokines. Whether this is a result of RBM10 involvement in splicing regulation, or in RIG-I/DENV genome ribonucleoprotein complex, or both, remains an intriguing question. The fact is that higher protein levels of RBM10 correlate with an anti-viral, pro-inflammatory scenario while lower levels with a pro-viral, anti-inflammatory one.

Fig 3: RBM10 binds RIG-I and DENV genome and promotes RIG-I ubiquitination. (A) RBM10 and dsRNA (a marker of viral RNA) localization was visualized by indirect immunofluorescence and nuclei-cytoplasmic ratio was quantified. (B) A549 cells were either treated with IFN α or DENV infected and subjected to co-immunoprecipitation assays with anti-RBM10. RNase A (0.1 mg/ml) was added to lysates when indicated. Input and immunoprecipitated fractions were analyzed by western blot with the corresponding antibodies. (C) RNA-immunoprecipitation (RIP) assays with anti-RBM10 of lysates from A549 infected cells at 48 hpi. RT-qPCRs were performed with primers for SAT1 pre-mRNA and viral RNA and represented as RIP/input RNA. Average values from triplicates are shown with standard deviation and p-values, determined using a paired two-tailed t-test. Significant P-values are indicated by the asterisks above the graphs (***P< 0.001; **P< 0.01; *P< 0.05). (D) HEK 293T were transfected with the indicated expression vectors and after 48 h subjected to Ni-NTA affinity purification. Eluates and input fractions were analyzed by western blot with the corresponding antibodies.

Fig 4: Genetic identification of the novel splice variant (NM_005676: c.17+1G>C, p.?) in RBM10. The pedigree of the family with the TARP-affected child (IP), the healthy father (HF) and the healthy carrier mother (CM). Familiar genotyping analyses of the RBM10-associated splice variant (NM_005676: c.17+1G>C, p.?) confirmed that the splice variant was inherited by the IP from the heterozygous CM, while the HF presented the normal reference allele. The sequencing peaks showing the nucleotide position of the splice variant is highlighted by a red frame and the mutated allele is indicated by an asterisk.

Fig 5: RBM10 is involved in the RLR signaling pathway. (A) A549 cells were transfected with the indicated siRNAs (40 nM) for 48 h, then mock treated (DMSO) or treated with 1 μM JAK inhibitor for additional 24 h during which, the last 9 h included Poly I:C intracellular administration. RNA was prepared and analyzed by RT-qPCR for IFN β. (B and C) A549 cells were transfected with the indicated siRNAs (40 nM) for 24 h and then treated with IFN α (50 000 U/ml) for additional 24 h. ISG15 (B) and IL-8 (C) mRNA levels were analyzed by RT-qPCR. Average values from triplicates are shown with standard deviation and P-values, measured with a paired two-tailed t-test. Significant P-values are indicated by the asterisks above the graphs (***P< 0.001; **P< 0.01; *P< 0.05).