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. Author manuscript; available in PMC: 2016 Nov 27.
Published in final edited form as: Nat Microbiol. 2016 May 27;2016:16069. doi: 10.1038/nmicrobiol.2016.69

Figure 6. Nuclear Speckle Assembly Factor SON Interacts with M1 mRNA and Mediators of M1 mRNA Splicing.

Figure 6

(a) VERO cells were transfected with control siRNA, Aly/REF siRNA, or UAP56 siRNA. After 48h, cells were infected with WSN (a) or WSN ΔNS1(b) at MOI 10 for 6h. Cells were then fixed for M mRNA labeling by RNA-FISH. Nuclear speckles were marked with SON antibody. Insets are enlargement of areas showing nuclear speckles. Images shown in a and b are representative of three independent experiments. Scale bar = 10µm. (c) Accumulation of M mRNA at nuclear speckles was quantified and compared between WSN and WSN ΔNS1 infected cells transfected with siRNA as in a and b. Values are mean ± s.d. of at least 11 cells that were analyzed in each treatment. p.i.- post infection. (d) RT-qPCR quantification of M2/M1 mRNA ratio in VERO cells transfected with control siRNA, Aly/REF siRNA, or UAP56 siRNA and infected for 6h with WSN or WSN ΔNS1 at MOI 1. The average M2/M1 mRNA ratio from control siRNA transfected cells infected with WSN (M2/M1 mRNA = 0.495) was set to 1 and the relative average M2/M1 ratio at the depicted conditions are shown. Values are mean ± s.d. from three independent experiments. ** T test p value < 0.01. (e–g) Cell extracts from uninfected or infected A549 cells treated with RNase inhibitor (RNasin) or with RNase A were subjected to immunoprecipitations with antibodies specific to NS1-BP or SON. Immunoprecipitates (Bound) and unbound fractions were subjected to western blot using antibodies specific to the depicted proteins. (h–i) SON binds directly to M1 mRNA. The entire M1 mRNA was radiolabeled uniformly at C residues, incubated with JSL1 nuclear extract under splicing conditions, crosslinked with 254 nm light and digested with RNase. The reaction was then either resolved by SDS-PAGE (25% Total) or incubated in separate reactions with the antibodies indicated (IP: anti-). Total reaction or immunoprecipitated proteins were resolved by SDS-PAGE. (j) Model for influenza virus M1 mRNA trafficking and splicing. Data shown in e–i are representative of three independent experiments.