Similarity of SF1 and QKI and RNA-seq analysis of SF1 or QKI loss-of-function in HepG2 cells.

A. Overlay of SF1 (cyan) and QKI (tan) KH and QUA2 protein domains: green highlighted region indicates KH and QUA2 domains of QKI; the two tan alpha helices on the left side make up the QUA1 domain in QKI (which SF1 lacks; text indicates residues in unstructured regions of QKI). B. Vast-tools analysis of the ENCODE RNA-seq data from SF1 (shSF1) or QKI shRNA (shQKI) knockdown compared to control shRNA (shNT) in HepG2 cells; the y-axis shows dPSI for shQKI or shSF1 relative to shNT for significantly altered alternatively spliced exons (AltEX; left) or intron retention (IR; right; dPSI ≥ |10| and MVdPSI > 0), and ****P < 0.0001 by Mann-Whitney U when comparing the distribution of changes in shQKI relative to shNT to shSF1 relative to shNT). C. Scatter plot showing the distribution of AltEX events that changed under depletion of both SF1 (dPSI values relative to shNT on the y-axis) and QKI (dPSI values relative to shNT on the x-axis); the number in each quadrant indicates how many AltEX events were observed; RAI14 exon 11 is shown in cyan. D. Simple Enrichment Analysis (SEA) of the intron region spanning 60 nt to 20 nt upstream of the 3’ss in QKI and SF1 regulated AltEX events shown in C (P < 0.05). E. rMAPS motif map for UACUAA generated for AltEX events changing during shSF1 compared to shNT by rMATS; motif scores (solid line) or -log10 P-value (dotted line) is shown in red for exons whose inclusion increases and in blue for exons whose inclusion decreases. F. rMAPS motif map as described in E. but for shQKI treatment compared to shNT.

RAI14 exon 11 is repressed by QKI and activated by SF1.

A. UCSC Genome Browser screenshot (top panel) with RNA-seq reads mapping to RAI14 for shQKI (top), shNT (middle), or shSF1 (bottom); percent spliced in (PSI) values are measured by Vast-tools; the inset shows boxed regions of (from top) two ACUAAC elements with Cola-seq branchpoints mapping to one nucleotide downstream of a branchpoint adenine, ACUAA elements by oligomatch, then QKI eCLIP peaks from K562 (top) or HepG2 (bottom) cells that overlap with these; conservation of 100 vertebrates is shown at the bottom. B. Western blot of protein extracted from HEK293 QKI KO cells (top) probed with anti-tubulin (magenta) or anti-panQKI (green) antibodies; RT-PCR of RAI14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the cells above showing mean percent included ± standard deviation below (n = 3 biological replicates; ****P < 0.001 by Student’s t-test compared to ctrl). C. Western blot (top) of proteins extracted from HEK293 cells transfected with siNT, siSF1_2 or siSF1_1+2, probed with anti-SF1 (green) or anti-Gapdh (magenta) antibodies; the protein abundance (fold change relative to the siNT control ± standard deviation) is shown below; RT-PCR of RAI14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the cells described above with mean percent included ± standard deviation below (n = 3 biological replicates; **P < 0.01 or ***P < 0.001 by Student’s t-test compared to siNT). D. Western blot (top) of proteins extracted from C2C12 myoblasts transfected with siNT, siQki or siSf1_1+2, probed with anti-SF1 (magenta; top), anti-tubulin (magenta; middle) or anti-panQki (green; bottom). The protein abundance (fold change relative to the siNT control ± standard deviation is shown below (n = 3 biological replicates; **P < 0.01 or ***P < 0.001 by Student’s t-test); RT-PCR of Rai14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the C2C12 cells described above with mean percent included ± standard deviation indicated below (****P < 0.0001 by Student’s t-test).

Analysis of DUP-RAI14 exon 11 (ex 11) splicing reporter.

A. Schematic of the beta globin pDUP-RAI14 ex 11 splicing reporters indicating the region of intron 10, exon 11, and intron 11 included; the inset below show the different constructs (wild type or mutant) tested. B. RT-PCR and BioAnalyzer gel-like image from RNA extracted from C2C12 cells transfected with RAI14 ex 11 wild type or deletion mutant reporters; mean percent included values are shown below (+/- the standard deviation; *indicates an unidentified/spurious product). C. RT-qPCR measuring total reporter RNA level, normalized to Eef1a1, from RNA described in B., and shown as fold-change relative to WT (**P < 0.01 or ****P < 0.001 by Student’s t-test). D. RT-PCR and BioAnalyzer gel-like image from RNA extracted from C2C12 cells transfected with RAI14 ex 11 wild type or substitution mutant reporters, analyzed as described in B. E. RT-qPCR measuring total reporter RNA level, normalized to Eef1a1, from RNA described in D., and shown as fold-change relative to WT (*P < 0.05 or ****P < 0.001 by Student’s t- test). F. RT-PCR and BioAnalyzer gel-like image from RNA extracted from C2C12 cells transfected with RAI14 ex 11 wild type or half-site mutant reporters, analyzed as described in B (***P < 0.001, ****P < 0.0001). G. RT-qPCR measuring total reporter RNA level, normalized to Eef1a1, from RNA described in F., and shown as fold-change relative to WT (**P < 0.01 or ****P < 0.001 by Student’s t-test). Each experiment was conducted in biological triplicate, and for RT-PCR with BioAnalyzer measurement, each comparison compared to WT reporter showed P < 0.0001 by Student’s t-test.

Analysis of the proteins associating with RAI14 intron 10 RNA.

RNA affinity chromatography (RAC) with liquid chromatography and tandem mass spec or western blot analysis. A. Schematic representation of substrates used for RNA affinity chromatography (RAC) which included 64 nt of RAI14 intron 10, 6 nt of exon sequence, and the tobramycin aptamer (tobra): wild type, upDEL, dnDEL, 2xDEL and aptamer only; C2C12 nuclear extract (NE) was incubated with these, and RAC-associated eluates were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or western blot. B. Heatmap showing hierarchical clustering of early spliceosome and 17S U2 snRNP protein (E/U2) abundance detected in the RAC-LC-MS/MS datasets for each substate shown and in the presence of ATP; these represent background-corrected levels relative to NE (see Methods) and the scale bar shows row Z-score values. C. Numbers observed for relative levels of E/U2 proteins (background corrected relative to NE) observed associating with each RAC substate with either a positive (npos) or negative (nneg) value. D. Western blot of NE (input) or WT, dnDEL (dn), or 2xDEL (2x) RAC time-course (+ATP as described in B and C) for 7.5 minutes (left), 15 minutes (middle), or 30 minutes (right) probed with anti-panQki (top) or anti-SF1(bottom) antibodies. E. Heatmap showing hierarchical clustering of early spliceosome and 17S U2 snRNP protein (E/U2) abundance detected in the RAC-LC-MS/MS datasets for each substate shown and in the absence of ATP; these represent data-independent acquisition (DIA; see methods) values normalized to NE and each mutant is shown as log2 fold change relative to the WT substate and passed cutoff of log2 fold change > |0.2| and P < 0.01. F. Volcano plot comparing LC-MS/MS log2 protein abundance (log2 fold-change (log2FC); x-axis) of E/U2 (circles) and other RBPs (squares) observed associating with RAC substates dnDEL compared to WT (y-axis shows -log10 P-value) of enriched proteins (cutoff: L2FC > |0.7| and P < 0.01); inset shows the number observed for those enriched in dnDEL (yellow) or WT (blue); schematic below shows model of RAC substates recruitment to distinct protein-associated species. G. Western blot of NE (input) or WT, dnDEL (dn), or 2xDEL (2x) RAC time-course (-ATP as described in E and F) for 7.5 minutes (left), 15 minutes (middle), or 30 minutes (right) probed with anti-panQki (top), anti-SF1 (middle), or anti-TATSF1 (bottom).

QKI5 expression is lethal in yeast and represses splicing.

A. BY4741 S. cerevisiae strain with EGFP (top), mutant QKI5 (mtQKI5; middle), or wild type QKI5 (wtQKI5; bottom) GAL-inducible transgene cultures grown on either glucose-(top) or galactose-containing (bottom) YPD plates at decreasing densities (left to right) and incubated at 30°C for 72h. B. Changes in RNA abundance measured by Deseq2 analysis of RNA-seq data for intronless, spliced, or unspliced transcripts were measured from RNA extracted from BY4741 cells with QKI5 expression (induced by galactose-containing media for 4h), or in the BY4147 parental strain also cultured in galactose-containing media for 4h (control), and are shown as L2FC in QKI5-induced cells relative to control (n = 3 biological replicates; cutoff P < 0.1; abundance cutoff of TPM > 0.2); **P < 0.01 or ****P < 0.0001). C. Splicing changes measuring the change in percent unspliced (Δ%Unspliced; y-axis) for BY4741 as described in B.; cutoff P < 0.1 by Student’s t-test and base mean > 100. D. RT-PCR with primers that span exon-intron-exon junctions and BioAnalyzer gel-like image showing mean percent unspliced (+/- SD; n = 3) below for introns whose inclusion increased upon QKI5 ectopic expression and as measured by RNA-seq analysis in C. for the parental or QKI5- expressing cells (**P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test). E. RT-qPCR analysis measuring mean percent unspliced transcript from RNA extracted from biological triplicate cultures of either the parental control or BY4741 expressing QKI5 for each transcript shown (+/- SD; *P < 0.05, ***P < 0.001, ****P < 0.001). F. Bar graph showing -log10 P-values (y-axis) of significantly enriched (SEA; P < 0.01) motifs observed in 3’ proximal ends of introns whose inclusion increases upon ectopic QKI5 expression in yeast (x-axis). G. UCSC Genome Browser screen shot showing RNA-seq reads mapping to the ACT1 transcript intron/exon junction near the 3’ss from RNA extracted from parental control and QKI5-induced cells; boxed sequences show two TACTAA elements.

Western blot and RT-PCR of proteins and RNA extracted from WT HEK293 cells transfected with tdTomato, WT myc:Qki5, MT myc:Qki5 and SF1.

The top panel shows a western blot probed with anti-SF1 (magenta), anti-PanQKI (green, middle) and anti-Gapdh (magenta, bottom). Below RT-PCR products analyzed on a Bioanalyzer from RNA extracted from transfected WT HEK 293 cells with mean percent included and ± standard deviation bellow (**P < 0.01,***P < 0.001 by Student’s t-test). The results shown are representative of 3 biological replicates.

Agarose gel showing PCR amplification in the absence (-) or presence (+) of reverse transcriptase.

PCR was performed with RNA from C2C12 cells transfected with RAI14 reporter plasmids.

Uncropped western blots from Fig 4.

A. Shows uncropped western blot image that is used to construct Fig 4D; QKI is shown in green and SF1 in red and the boxes indicate the cropped region. B.

A. Growth curve of Gal-inducible EGFP, mtQKI5 and WT QKI5 BY4741 yeast cells grown in the absence (blue) or presence of galactose (orange). The y-axis represents the log2 number of cells, and the horizontal axis indicates the time point (in hours) at which the cells were collected and counted. B. Representative phase contrast microscopy showing images of EGFP, mtQKI5 and WT QKI5 expressing yeast cells (1000x) at 24 hours after galactose induction. The inset shows enlarged regions to provide more detailed information on cell morphology. C. RT-PCR of parental BY4741 or BY4741 with the QKI5 transgene 4h after galactose induction, measuring various intron-retention events predicted upon ectopic QKI5 expression (with exception of control HAC1) using primers that span intron-exon junction for each target and analyzed on an agarose gel (n = 3 per condition).