Competition for the conserved branch point sequence influences physiological outcomes in pre-mRNA splicing
- Transplant Division, Department of Surgery, University of Texas Medical Branch, United States
- RNA Genomics and Structure, Genome Institute of Singapore, Agency for Science, Technology, and Research (A*STAR) Singapore, Singapore
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, United States
- Sinsheimer Labs, RNA Center for Molecular Biology, Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, United States
Figures
Figure 1
Similarity of Splicing Factor 1 (SF1) and Quaking (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.
Figure 2 with 1 supplement
RAI14 exon 11 is repressed by QKI and activated by Splicing Factor 1 (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 Quaking (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 (MW in kD is shown on right); 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; size of amplicon in bp is shown on right). (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 and MW in kD is shown on right; 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; size of amplicon in bp is shown on right). (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); MW in kD is shown on right. 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; size of amplicon in bp is shown on right).
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Figure 2—source data 1
Original membranes and BioAnalyzer gel-like images corresponding to Figure 2A (left), Figure 2C (middle), or Figure 2D (right).
See text under each set of images for additional details.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig2-data1-v1.pdf
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Figure 2—source data 2
Source data containing original uncropped western blots and BioAnalyzer gel-like images/files.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig2-data2-v1.zip
Figure 2—figure supplement 1
Western blot and RT-PCR of proteins and RNA extracted from wild-type (WT) HEK293 cells transfected with tdTomato, WT myc:Qki5, MT myc:Qki5, and Splicing Factor 1 (SF1).
The top panel shows a western blot probed with anti-SF1 (magenta), anti-PanQKI (green, middle), and anti-Gapdh (magenta, bottom); MW in kD is shown to the right. Below RT-PCR products analyzed on a Bioanalyzer from RNA extracted from transfected WT HEK 293 cells with mean percent included and ± standard deviation below (**p<0.01,***p<0.001 by Student’s t-test). The results shown are representative of three biological replicates; size in bp is shown to the right.
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Figure 2—figure supplement 1—source data 1
Original uncropped membrane from western blot and uncropped gel-like image from BioAnalyzer shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Original uncropped membrane from western blot and uncropped gel-like image from BioAnalyzer shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
Analysis of the 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 shows 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.
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Figure 3—source data 1
Original BioAnalyzer gel-like images corresponding to Figure 3B (left), Figure 3D (middle), or Figure 3F (right).
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig3-data1-v1.pdf
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Figure 3—source data 2
Original BioAnalyzer gel-like images and data files corresponding to Figure 3B (left), Figure 3D (middle), or Figure 3F (right).
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig3-data2-v1.zip
Figure 3—figure supplement 1
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.
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Figure 3—figure supplement 1—source data 1
Original uncropped agarose gel images from Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Original uncropped agarose gel images from Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig3-figsupp1-data2-v1.zip
Figure 4
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 (2 x) RAC time-course (+ATP as described in B and C) for 7.5 min (left), 15 min (middle), or 30 min (right) probed with anti-panQki (top) or anti-SF1 (bottom) antibodies; MW shown in kD to the right. (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 RNA-binding proteins (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 (2 x) RAC time-course (-ATP as described in E and F) for 7.5 min (left), 15 min (middle), or 30 min (right) probed with anti-panQki (top), anti-SF1 (middle), or anti-TATSF1 (bottom); MW shown in kD to the right.
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Figure 4—source data 1
Original membranes corresponding to western blots depicted in Figure 4D (left), or Figure 4G.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig4-data1-v1.pdf
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Figure 4—source data 2
Original membranes corresponding to western blots depicted in Figure 4D (left), or Figure 4G.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig4-data2-v1.zip
Figure 5 with 1 supplement
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 72 hr. (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 4 hr), or in the BY4147 parental strain also cultured in galactose-containing media for 4 hr (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); amplicon size in bp is shown to the left for each except YDL012C, which is shown to the right. (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 screenshot 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.
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Figure 5—source data 1
Original gel-like images from BioAnalyzer used in Figure 5D.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig5-data1-v1.pdf
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Figure 5—source data 2
Original gel-like images and data file from BioAnalyzer used in Figure 5D.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig5-data2-v1.zip
Figure 5—figure supplement 1
QKI5 expression is lethal in yeast and represses splicing.
(A) Growth curve of Gal-inducible EGFP, mtQKI5, and wild-type (WT) QKI5 BY4741 yeast cells grown in the absence (blue) or presence of galactose (orange). The y-axis shows the log2 number of cells, and the x-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 (1000 x) at 24 hr 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 4 hr after galactose induction, measuring various intron-retention events predicted upon ectopic QKI5 expression (with the exception of control HAC1) using primers that span intron-exon junction for each target and analyzed on an agarose gel (n=3 per condition).
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Figure 5—figure supplement 1—source data 1
Original uncropped agarose gel images of data shown in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig5-figsupp1-data1-v1.pdf
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Figure 5—figure supplement 1—source data 2
Original uncropped agarose gel images of data shown in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/103167/elife-103167-fig5-figsupp1-data2-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (yeast) | BY4741 | Ares Lab/Prakash Lab | NCBI Taxonomy ID: 1247190 | |
| Cell line (mouse) | C2C12 | ATCC | CRL-1772 | |
| Cell line (human) | Flp-In T-Rex 293; WT or QKI KO | Thermo Fisher | RRID:CVCL_U427 | |
| Transfected construct (human) | pDUP-RAI14 (WT and various mutants) | Parental plasmid Dominski and Kole, 1991 | ||
| Transfected construct (mouse) | pMyc:Qk5 | Sean Ryder | ‘Wild-type’ but contains a translationally silent mutation in the region corresponding to exon 6 | |
| Transfected construct (mouse) | pMyc:Qk5 (K120A;R124A) | Fagg et al., 2022 | QKI5 defective in RNA binding | |
| Transfected construct (human) | pcDNA3.1-SF1 | This paper | See ‘Plasmids and Transfections’ section in Methods below | |
| Other | pcDNA3.1-tdTomato | Fagg et al., 2017 | Fagg et al., 2017 | |
| Other | pJW1666 | Addgene plasmid # 112040 | Jonathan Weissman (Addgene plasmid # 112040) | |
| Other | pJW1666-Qki5 | Addgene plasmid # 112040 with Qki5 inserted | Jonathan Weissman (Addgene plasmid # 112040) with Qki5 inserted | |
| Other | pJW1666-Qki5 (K120A;R124A) | Addgene plasmid # 112040 with Qki5 (K120A;R124A) inserted | Jonathan Weissman (Addgene plasmid # 112040) with Qki5 (K120A;R124A) inserted | |
| Recombinant DNA reagent | pcDNA5-aptamer plasmid | Garcia-Blanco lab | ||
| Antibody | GAPDH; Mouse monoclonal IgM | Sigma | G8795 | 1:40,000 |
| Antibody | α/β-Tubulin; Rabbit polyclonal | Cell Signalling | 2148 | 1:5000 |
| Antibody | panQKI; Mouse monoclonal IgG2b (clone N147/6) | Sigma | MABN624 | 1:2000 |
| Antibody | SF1; Rabbit polyclonal | Bethyl | A303-213a | 1:5000 |
| Antibody | TATSF1; Rabbit polyclonal | Thermofisher | 20805–1-AP | 1:2000 |
| Antibody | IRDye 800CW Goat anti-Mouse IgG2b; Secondary anti-mouse | Licor | P/N 926–32352 | 1:15,000 |
| Antibody | IRDye 800CW Goat anti-Rabbit IgG Secondary anti-rabbit | Licor | P/N: 926–32211 | 1:15,000 |
| Antibody | IRDye 680RD Goat anti-Mouse IgM Secondary anti-mouse | Licor | P/N: 926–68180 | 1:20,000 |
| Antibody | IRDye 680RD Goat anti-Rabbit IgG Secondary anti-rabbit | Licor | P/N: 926–68071 | 1:20,000 |
| Commercial assay or kit | SYBR Green Universal Master Mix | Applied Biosystems | ||
| Commercial assay or kit | Taq2x Master Mix | New England Biolabs | ||
| Commercial assay or kit | ReliaPrep RNA mini prep kit | Promega | ||
| Commercial assay or kit | iScript Reverse Transcription Supermix | BioRad | ||
| Commercial assay or kit | NEBNext poly(A) mRNA Magnetic Isolation module | New England Biolabs | NEB, E7490 | |
| Commercial assay or kit | NEBNext Ultra II Directional RNA Library Prep kit for Illumina | New England Biolabs | NEB, E7760 | |
| Commercial assay or kit | HiScribe T7 High Yield RNA Kit | New England Biolabs | NEB, E2040 | |
| Software, algorithm | UCSF ChimeraX | https://www.rbvi.ucsf.edu/chimerax | RRID:SCR_015872 | Version 1.10.1 |
| Software, algorithm | Vast-tools | Irimia et al., 2014; Tapial et al., 2017 | 2.0.2, database has16.02.18 | |
| Software, algorithm | rMATS | Shen et al., 2014; Park et al., 2016 | Version 4.0.2, GENCODE v30 annotation | |
| Software, algorithm | rMAPS2 | rMAPS2; https://rmaps.cecsresearch.org/ | ||
| Software, algorithm | Trimmomatic | Trimmomatic | RRID:SCR_011848 | v.0.39 |
| Software, algorithm | Kallisto | Kallisto | RRID:SCR_016582 | v.0.50 |
| Software, algorithm | Deseq2 | Love et al., 2023 | v1.42.1 | |
| Software, algorithm | Simple Enrichment Analysis (SEA) | Bailey and Grant, 2021; https://meme-suite.org/meme/doc/sea.html | ||
| Software, algorithm | XCalibur | Thermo Scientific | RRID:SCR_014593 | V2.5 |
| Software, algorithm | Proteome Discoverer | Thermo Fisher | RRID:SCR_014477 | Version 2.2.0388 |
| Software, algorithm | Scaffold | Proteome Software Inc. | RRID:SCR_014321 | Version Scaffold_4.11.1 |
| Software, algorithm | Protein Prophet | Nesvizhskii et al., 2003 | ||
| Software, algorithm | MSConvert | Chambers et al., 2012 | ||
| Software, algorithm | MSFragger | Kong et al., 2017 | ||
| Software, algorithm | DIA-NN | https://github.com/vdemichev/DiaNN; Demichev et al., 2020 | ||
| Software, algorithm | Fragpipe-Analyst | Hsiao et al., 2024 | ||
| Sequence-based reagent | Oligonucleotides and RNAi | See Supplementary file 8 |
Additional files
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Supplementary file 1
Vast-tools splicing analysis datafile from ENCODE RNA-seq data.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp1-v1.xlsx
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Supplementary file 2
rMATS splicing analysis datafile from ENCODE RNA-seq data comparing shQKI to shControl.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp2-v1.xlsx
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Supplementary file 3
rMATS splicing analysis datafile from ENCODE RNA-seq data comparing shSF1 to shControl.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp3-v1.xlsx
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Supplementary file 4
LC-MS/MS datafile from RNA affinity chromatography in the presence of ATP.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp4-v1.xlsx
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Supplementary file 5
LC-MS/MS datafile from RNA affinity chromatography in the absence of ATP.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp5-v1.xlsx
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Supplementary file 6
Transcript abundance analysis of RNA-seq data from BY4741 with QKI5 expression compared to parental control.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp6-v1.xlsx
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Supplementary file 7
Splicing analysis of RNA-seq data from BY4741 with QKI5 expression compared to parental control.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp7-v1.xlsx
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Supplementary file 8
Oligonucleotide sequence info file.
- https://cdn.elifesciences.org/articles/103167/elife-103167-supp8-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/103167/elife-103167-mdarchecklist1-v1.docx
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Competition for the conserved branch point sequence influences physiological outcomes in pre-mRNA splicing
eLife 13:RP103167.
https://doi.org/10.7554/eLife.103167.3
