Fig 2: Viral and host models for hnRNP K- and NS1-BP-regulated splicing. a Model of hnRNP K and NS1-BP splicing regulation of influenza M segment splicing (as described in text). b Putative model of hnRNP K and NS1-BP regulated splicing of host RNA

Fig 3: Host proteins hnRNP K and NS1-BP regulate IAV PR8 M segment splicing. a Model for hnRNP K and NS1-BP regulated M segment splicing based on previous studies6,7. b Immunoblots of A549 whole cell extract 12 h post PR8 infection in the context of mock, hnRNP K or NS1-BP siRNA knockdown. Molecular weights are indicated to left of each blot. c Diagram of IAV PR8 M segment mRNA with possible splice isoforms and extension primer positions labeled. Boxes denote exons and the line denotes an intron. M3 and M2 indicate potential 5'ss and numbers indicate nucleotide position. d Primer extension of PR8 M segment antisense RNA after 12 h infection in A549 cells at moi = 2. Cells were mock transfected or treated with 50 nM siRNA targeted at hnRNP K or NS1-BP. Extension primers used are indicated on the left and band identities on the right of the gel image. Uncropped gels shown in Supplementary Fig. 5. e Quantification of mRNA primer extension products represented as percentage signal of each isoform relative to signal of total isoforms (M1 + M2 + M3). Values are means ± s.d. from three independent experiments. For all experiments in manuscript, a minimum of 3 experiments were performed to insure appropriateness of statistical tests. Statistical significance was determined via two-tailed student’s t test, where *p < 0.1 and **p < 0.05 when comparing mock vs siRNA samples

Fig 4: Mutation of hnRNP K but not NS1-BP-binding sites alters M mRNA localization. a A549 cells were infected with wild type (WT) and mutant A/WNS/33 viruses at MOI 5. After 8.45 h infection, cells were subjected to RNA-FISH combined with immunofluorescence to detect M mRNA and SON protein. SON protein is a marker for nuclear speckles. Scale bar, 20 µm. The marked rectangular region is enlarged and shown in the right-most panel. Scale bar, 5 µm. Images are representative of at least 12 images from biological triplicates. b Quantification of M mRNA intensity in the cytoplasm (C) and in the nucleus (N) expressed as C/N ratios. Values are mean ± s.d. measured in at least 25 cells. c Quantification of M mRNA intensity at nuclear speckles with respect to total cell intensity. Values are mean ± s.d. measured in at least 25 cells. Statistical significance was determined via two-tailed student’s t test, where **p < 0.01 and ****p < 0.001 when comparing mock vs. mutant samples

Fig 5: hnRNP K binding site promotes U1 snRNP binding. a Diagram of in vitro transcribed, biotinylated RNA affinity baits derived from IAV M segment mRNA. Blue text corresponds to NS1-BP binding, red text corresponds to hnRNP K binding, based on data from Fig. 2. Grey text corresponds to mutated nucleotides. b RNA affinity purification of proteins bound to baits: 1–106 WT, 1–106 pY-mut, 1–106 pC-mut, 1–106 pCpY-mut. RNA probes were incubated in JSL1 nuclear extract and isolated with streptavidin beads. Bead eluates were resolved via SDS-PAGE and either silver stained (top panel) or immunoblotted for specific proteins (bottom panels). Molecular weights are indicated to left of each blot. c Diagram of in vitro transcribed AMT psoralen cross-linking substrates derived from IAV M segment mRNA. Position of extension primer is shown below. d Radiolabeled primer extension of M segment RNA substrates after AMT psoralen cross-linking. RNA substrates were incubated with JSL1 nuclear extract ± presence of U1 snRNP, supplemented with AMT psoralen, and irradiated with 365 nm UV light. Cross-link products were visualized by primer extension. Identity of cross-link products was determined using dideoxy sequencing (Supplementary Fig. 3). M2 U1 crosslinking quantification is in arbitrary units representing the signal of bands at the M2 5´ss in the presence of U1 ( + U1) divided by the sum of bands in the presence and absence of U1. Values are means ± s.d. from three independent experiments. Uncropped gels shown in Supplementary Fig. 5