Figure 5: FMRP KH2 domain mediates NP binding and is critical for stimulating RNA synthesis and viral RNP assembly.

(a) Schematic of Flag-tagged FMRP truncation mutants. (b) FMRP interacts with NP via the KH2 domain. Flag-tagged FMRP truncation mutant plasmids were individually transfected into 293T cells together with TAP-NP plasmid. After 36 h, cell lysates were prepared followed by TAP purification and western blot analyses. (c) FMRP KH2 domain is critical for stimulating viral RNA synthesis. 293T cells were transfected with plasmids expressing the HK68 RNP reconstitution plasmids together with Flag-tagged full-length FMRP or FMRP truncation mutant plasmids. Total RNAs were extracted and levels of mRNA, cRNA and vRNA were detected by primer extension analyses. (d) Statistical analysis of viral RNAs in (c). Values of viral RNAs in cells transfected with full-length FMRP or FMRP mutants were standardized to the 5S rRNA level and then normalized to levels of viral RNAs in cells transfected with a control vector (mean±s.d. of three independent experiments, *P<0.05, **P<0.01 and ***P<0.001, two-tailed Student’s t-test). (e) The FMRP KH2 domain is critical for stimulating viral RNP assembly. 293T cells were transfected with plasmids expressing PB1-TAP, PA, PB2 and NP, with or without the viral RNA expression plasmid, along with increasing amounts of the FMRP or FMRP-ΔKH2 plasmid. After 36 h, cell lysates were prepared followed by TAP purification and western blot analyses (mean±s.d. of three independent experiments, *P<0.05, two-tailed Student’s t-test). (f) A point mutation (I304N) in the KH2 domain of FMRP disrupts FMRP–NP association. 293T cells were transfected with Flag-tagged WT FMRP or FMRP I304N mutant expression plasmids together with TAP-NP. After 36 h, cell lysates were prepared followed by TAP purification and western blot analyses. (g) The FMRP I304N mutant is defective in stimulating viral RNA synthesis. 293T cells were transfected with Flag-tagged WT FMRP or FMRP (I304N) plasmids together with the HK68 RNP reconstitution system. Total RNAs were prepared and analysed as indicated in (c).