Fig 2: RBM39 interactions with spliceosome components. (A) RBM39 interacts with U1 snRNP and U2AF. Interaction of RBM39 with the U1-specific protein U1-70K, the U2-specific protein U2A’ and the small subunit of U2AF was assayed by immunoprecipitations. HeLa cells were transiently transfected with U1-70K-GFP, U2A'-GFP or U2AF35-GFP, immunoprecipitated with anti-GFP antibodies and probed with antibodies shown to the right. U1C and SF3B4 served as positive controls for immunoprecipitations for U1-70K-GFP and U2A’-GFP, respectively. Asterisks denote a partially degraded U2A’-GFP. (B, C) RBM39 interactions monitored by FRET. Cells were transiently co-transfected with RBM39-CFP and C-terminally YFP-tagged U1-70K. (B) YFP was bleached in a small region comprising the nucleoplasm and nuclear speckles; CFP fluorescence was measured before and after bleaching. Fluorescence of RBM39 increased after bleaching of U1-70K-YFP [cf. CFP fluorescence in the bleached region (rectangles) before (top panel) and after (bottom panel) bleaching]. A, acceptor; D, donor; scale bar, 5 µm. (C) Quantification of individual donor-acceptor FRET efficiencies upon the inhibition of RNA polymerase II by DRB. Columns indicate means; errors bars SEMs. Interaction between RBM39-CFP and U2AF35-YFP (22) served as a positive control and interaction between RBM39-CFP and YFP as a negative control. Significantly different means are denoted by an asterisk (P< 0.01; t-test).

Fig 3: Alternative splicing of hnRNP genes regulated by PUF60/RBM39. (A) HNRNPK. (B) HNRNPD. (C–F) RT-PCR validation. Down- and up-regulated exonic segments are marked by red and green rectangles at the top, respectively. The siRNA concentrations were 50 and 90 nM. SC, scrambled siRNA controls. Exons (e) targeted by amplification primers (Supplementary Table S1) are at the bottom. Columns show the relative abundance of the indicated transcripts (shown in panels A and B).

Fig 4: RNA-Seq of HEK293 cells depleted of PUF60 and RBM39. (A) Domain structure. (B) Western blot analysis of HEK293 cells lacking or overexpressing PUF60 (left panel) and lacking RBM39 (right panel). hd, homodimers; ex, exogenous; en, endogenous protein; sc, scrambled siRNA controls; EV, empty vector. (C) Distribution of RNA processing events altered by depletion of PUF60 and RBM39. Each event was confirmed in the genome browser by visualizing complete transcripts and cleaned APA sites annotated in the APA atlas (43).

Fig 5: Alternative splicing of U-binding interaction partners of PUF60 in depleted cells. (A–D) Genome browser views of RNA-Seq tracks in control (C) and depleted (–) cells. Down- and up-regulated exonic segments are marked by red and green rectangles at the top, respectively. Y-axis, sequencing read numbers. R1, R2; replicates. Peptides encoded by PUF60/RBM39-dependent exons are shown at the bottom together with their intron-proximal (P) or -distal (D) 3'ss. The 3'-seq tracts superimpose the APA atlas data (43). (A) TIAR. (B) TIA-1. (C) HNRNPC. (D) MATR3/SNHG4. (E–H) RT-PCR validation from independent transfections. The final siRNA concentrations were 50 and 90 nM. SC, scrambled controls. Exons (e) containing amplification primers (Supplementary Table S1) are to the left and RNA products are to the right in each panel. Columns show the relative abundance of the indicated transcripts (shown in panels A–D). (I, J) Immunoblotting of PUF60- and control cells with anti-TIAR (I) and anti-TIA-1 (J) antibodies. The extra band between TIA-1a and TIA-1b is likely to result from phosphorylated residue(s) reported in the peptide shown in panel B (http://www.phosphosite.org).