Alternative splicing regulation of cell-cycle genes by SPF45/SR140/CHERP complex controls cell proliferation

Abstract

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation, but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140, and CHERP, form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3' splice sites. These comprise alternative exons embedded in genes with important functions in cell-cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulates the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.

Keywords: CHERP; SPF45; SR140; alternative splicing; cell proliferation.

FIGURE 1.

SPF45, SR140, and CHERP cross-regulate each other at the protein level. (A) Similar splicing perturbation profiles upon knockdown of SPF45, SR140, or CHERP. Robust z-score values of changes in alternative splicing (y-axis, >0: inclusion <0: skipping) for 39 exons in the indicated genes (x-axis) were extracted from Papasaikas et al. (2015). The high similarity between the profiles of splicing changes of the three factors identified a functional module (right panel) in a systematic comparison across splicing factors (Papasaikas et al. 2015). (B,C) Western blot analyses of SPF45, SR140, CHERP, and DHX15 (and Tubulin/actin as loading controls) upon their individual siRNA-mediated knockdowns in HeLa and U2OS cells. The results shown are representative of three independent experiments. (D) Western blot analysis of interactors of SPF45, SR140, or CHERP (and tubulin as loading control) of the same samples as in B. The result shown is representative of two independent experiments. (E) RT-qPCR-measurements of the fold change of mRNA levels of SPF45, SR140, and CHERP upon their individual knockdowns in HeLa and U2OS cells compared to siCNT. Data are represented as mean ± SD of three biological replicates, and P-values are calculated by one-way ANOVA followed by Dunnett's multiple comparison test on ΔΔCt values ([*] P < 0.01; [**] P < 0.001, [***] P < 0.0001). (F) Western blot analysis of SPF45, SR140, and CHERP (and actin as a loading control) upon individual or combined overexpression of these factors in HeLa cells. A total of 250 ng, 500 ng, or 1 µg of DNA was transfected (in individual overexpression assays these amounts correspond to single plasmids, while in combined overexpression assays they correspond to the sum of equal amounts of plasmids). See also Supplemental Figure S1.

FIGURE 2.

SPF45, SR140, and CHERP form a functional physical complex. (A, upper panel) Western blot analysis of SPF45, SR140, and CHERP (and tubulin as loading control) upon 3′UTR-targeted siRNA knockdowns in Flp-In T-Rex HeLa cells stably overexpressing either an empty vector (pcDNA5), a SPF45 cDNA containing a hairpin to prevent translation (SPF45hp), or cDNAs of SPF45, SR140, or CHERP with 3′UTRs not targeted by the siRNAs utilized. Twenty-four hours after siRNA transfection, protein expression was induced using tetracycline, and total cell extracts were prepared 48 h later. The results shown are representative of three independent experiments. (Lower panel) Quantification of western blot results, represented as mean ± SD of three biological replicates. P-values calculated comparing to pcDNA5 by one-way ANOVA followed by Dunnett's multiple comparison test ([*] P < 0.01; [**] P < 0.001). (B) Quantification of MADD exon 16 AS by RT-PCR followed by capillary electrophoresis, represented as fold change compared to siCNT in the same experimental conditions of A. Data are represented as mean ± SD of three biological replicates, and P-values are calculated comparing to pcDNA5 by Student's t-test ([**] P < 0.01). (C) Immunoprecipitation of endogenous SPF45 or CHERP in total protein extracts from HeLa cells and detection of coimmunoprecipitating proteins by western blot analyses. (D) Fractionation of HeLa nuclear extract (NE) through a 5%–40% glycerol gradient followed by western blot analysis of the indicated proteins. The approximate size of the fractions was estimated by profiling a molecular weight marker kit through a parallel gradient and staining it with Coomassie. The result shown is representative of three independent experiments.

FIGURE 3.

The SPF45/SR140/CHERP complex is required for cell proliferation, cell-cycle progression, and inhibition of apoptosis. (A, left panels) Western blot analyses of SPF45, SR140, or CHERP (and tubulin as a loading control) upon their individual knockdowns using lentiviral transduction of shRNAs in HeLa and U2OS cells. The results shown are representative of three independent experiments, which are quantified in the right panels and represented as mean ± SD with P-values calculated comparing to shCNT by one-way ANOVA followed by Dunnett's multiple comparison test ([*] P < 0.05; [**] P < 0.001; [***] P < 0.0001). (B) Cell proliferation measured by direct cell number count of HeLa or U2OS cells upon lentivirus-mediated knockdown of SPF45, SR140, or CHERP measured 24, 48, 72, and 96 h after plating. Data are represented as mean ± SD of three biological replicates. (C, left) Representative clonogenic assays (left) of HeLa or U2OS cells upon lentivirus-mediated knockdown of SPF45, SR140, or CHERP. (C, right) Quantification of the number of colonies formed after seeding 1000 HeLa or 2000 U2OS cells, represented as mean ± SD of three biological replicates with P-values calculated comparing to shCNT by one-way ANOVA followed by Dunnett's multiple comparison test ([*] P < 0.01; [**] P < 0.001; [***] P < 0.0001). (D) Distribution of HeLa or U2OS cells in different phases of cell cycle upon lentivirus-mediated knockdown of SPF45, SR140, or CHERP, analyzed by propidium iodide (PI) staining and flow cytometry. Data are represented as mean ± SD of three biological replicates with P-values calculated comparing to shCNT by χ² test ([*] P < 0.01; [**] P < 0.001; [***] P < 0.0001). (E, upper panels) Western blot analysis of the indicated cell-cycle markers (and GAPDH as loading control) upon knockdown of SPF45, SR140, or CHERP or upon treatment with UV light (UV-C at 10 J/m² for 6 h) as a control of Chk1 phosphorylation. (Lower panels) Quantification of the results, represented as mean ± SD of three biological replicates.

FIGURE 4.

SPF45, SR140, and CHERP co-regulate a program of AS events in genes relevant for cell-cycle progression. (A) Summary of AS changes upon knockdown of SPF45, SR140, or CHERP in HeLa cells. Total RNA from duplicates of each condition was used for RNA-seq analysis and AS changes were quantified using VAST-TOOLS. The number of AS events (y-axis) displaying increased inclusion (ΔPSI ≥ 15%) or skipping (ΔPSI ≤ −15%) are indicated for each type of AS event (x-axis). (B) Validation of events detected by VAST-TOOLS in RNA-seq data sets using independent RNA samples and RT-PCR followed by capillary electrophoresis upon knockdown of SPF45, SR140, or CHERP in HeLa cells. RT-PCR PSI values (bars) and VAST-TOOLS PSI values from RNA-seq data sets (orange line) display an overall correlation (R²) of 0.98 (shCNT), 0.73 (shSPF45), 0.76 (shSR140), and 0.57 (shCHERP). Data are represented as mean ± SD for three biological replicates. (C) Pairwise correlations of PSI values from AS events (|ΔPSI| ≥ 15%) upon knockdown of SPF45, SR140, or CHERP. Pearson correlation coefficients are shown in the upper left corner. (D) Venn diagram of the overlap between differentially spliced cassette exons (CEx) upon knockdown of SPF45, SR140, and CHERP. (E) Matt analyses of splicing regulatory sequence features enrichment, GC content, and length of CEx more included (UP) or more skipped (DOWN) upon knockdown of SPF45, SR140, CHERP, or all three (Triple overlap) compared to a stratified control set of nonchanging alternative exons. Sequence features include 5′ss and 3′ss strengths (maximum entropy scores), best predicted branch point (BP) sequence score, and polypyrimidine tract (Py) sequence score. Normalized differences in the median of the scores of each feature between each condition and nonchanging alternative exons were tested by Mann–Whitney U-tests ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001). See also Supplemental Figure S4E,F. (F) GO analysis of alternatively spliced exons upon SPF45 knockdown in HeLa cells performed using GOrilla. In purple, “G2/M transition” categories. (G) Word cloud depicting the genes from the “G2/M transition” categories from F. The size of genes is proportional to the ΔPSI of the contained events in SPF45 knockdown samples and pink genes correspond to events regulated by the three knockdowns (triple overlap). See also Supplemental Figures S4, S5.

FIGURE 5.

SPF45/SR140/CHERP regulate FOXM1 AS. (A) Scheme of AS events involving FOXM1 exon 9 (upper panel) and their changes upon knockdown of SPF45, SR140, or CHERP measured by RT-PCR followed by capillary electrophoresis (lower panel). The isoform identity of the amplification products is indicated. (B) Validation of FOXM1 exon 9 AS events (solid bars: full exon inclusion; striped bars: shorter exon inclusion) changes measured as in A in HeLa and U2OS cells. Data are represented as mean ± SD of three biological replicates, and P-values are calculated comparing to shCNT by Student's t-test ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001). (C) Structural domain organization of FOXM1 isoforms. The different domains are indicated as well as the location and likely functional impact of inclusion of sequences corresponding to exon 6 and exon 9 inclusion. PTC: premature termination codon. (D) Fold change over shCNT of mRNA levels of FOXM1 isoforms and total FOXM1 quantified by RT-qPCR upon knockdown of SPF45, SR140, or CHERP in HeLa and U2OS cells. Data are represented as mean ± SD of three biological replicates, and P-values are calculated by comparing to shCNT by one-way ANOVA followed by Dunnett's multiple comparison test ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001). (E) Relative abundance of the indicated common or isoform-specific peptides in control versus SPF45-depleted cells by targeted proteomics, normalized by the levels of ACLY, and corresponding fold changes (in log₂) between the two conditions. (F) Relative expression levels of known targets of transcriptional activation by FOXM1, quantified by Cuffdiff in RNA-seq data sets of knockdown of SPF45, SR140, or CHERP in HeLa cells. Gene expression is displayed as FPKM values (y-axis) and significant values—according to Cuffdiff differential gene expression test—are indicated by an asterisk. See also Supplemental Figure S7.

FIGURE 6.

AS of the SPF45/SR140/CHERP target FOXM1 exon 9 influences cell proliferation. (A) Schematic diagram of antisense oligonucleotides (AONs) targeting FOXM1 exon 9 5′ss and alternative 5′ss to promote exon skipping. Upper/lower case indicates exonic/intronic nucleotides, respectively. (B, upper panels) FOXM1 exon 9 isoforms inclusion (PSI) upon FOXM1 AONs (50 nM) transfection after SPF45 knockdown, assessed by RT-PCR and capillary electrophoresis. AONs predicted not to target human sequences (control), targeting the alternative 5′ss of FOXM1 and an equimolar combination of AONs targeting the 5′ss and alternative 5′ss (total 50 nM) were used in HeLa and U2OS cells. (Lower panel) quantification of these results, represented as mean ± SD of four biological replicates; P-values were calculated by Student's t-test ([*] P < 0.05; [**] P < 0.01) comparing the PSI values to those for CNT AON within each condition. (C) Percentage of plated cells forming colonies (clonogenic capacity, y-axis) upon FOXM1 AONs (50 nM) transfections in HeLa and U2OS cells knocked down for SPF45. Data are represented as mean ± SD of four biological replicates, and P-values are calculated by one-way ANOVA followed by Dunnett's multiple comparison test (ns = not significant; [**] P < 0.001; [***] P < 0.0001). (D) Schematic representation of predicted base-pairing interactions for canonical U1 snRNA and for engineered U1 snRNAs (alt 5′ss U1, 5′ss U1, and shifted U1) with FOXM1 exon 9 alternative 5′ss, 5′ss and downstream intronic region. Bases in bold indicate sequences different from the canonical U1 sequence and black dots depict wobble base-pairings. (E) Quantification of FOXM1 exons 9 inclusion (PSI) upon transfection of wild type or variant U1 snRNAs, measured by RT-PCR and capillary electrophoresis in HeLa cells. Cells were cotransfected with a GFP plasmid to allow sorting of GPF-positive cells, which coexpress U1 snRNAs, 24 h after transfection. Data are represented as mean ± SD of three biological replicates, and P-values are calculated comparing to GFP by Student's t-test ([**] P < 0.01; [***] P < 0.001). (F) Clonogenic assay of GFP-positive HeLa cells sorted 24 h after cotransfection of GFP- and U1 snRNA-expressing plasmids. The number of colonies is normalized to that of control samples transfected only with the GFP plasmid (y-axis). Data are represented as mean ± SD of three biological replicates, and P-values are calculated by one-way ANOVA followed by Dunnett's multiple comparison test ([***] P < 0.0001). See also Supplemental Figure S7.

FIGURE 7.

AS of the SPF45/SR140/CHERP target SPDL1 exon 3 influences cell proliferation. (A) Expression levels of SPF45, SR140, and CHERP in a data set monitoring gene expression levels through two continuous cell division cycles of synchronized HeLa cells (Dominguez et al. 2016). Gene expression values are displayed as normalized FPKM (y-axis) throughout two cell cycles after double thymidine block (x-axis). (B) Splicing pattern of the 28 AS events regulated by SPF45, SR140, and CHERP that overlap with cycling events as defined by Dominguez et al. Splicing changes are quantified as scaled PSIs (y-axis) through two cell division cycles after double thymidine block (x-axis). Events are grouped in two clusters (dark and light gray lines) according to their cycling pattern; cluster 1 includes FOXM1 exon 9 (red line). (C) Schematic representation of the alternatively spliced exon 3 of the SPDL1 gene, which includes an in-frame premature termination codon (PTC) that down-regulates SPDL1 protein expression. (D) Increased inclusion of SPDL1 exon 3 upon knockdown of the indicated components of the SPF45/SR140/CHERP complex, measured by RT-PCR and gel electrophoresis (left panel), quantified in the right panel as mean ± SD of three technical replicates, and P-values are calculated by one-way ANOVA test ([***] P < 0.001). (E, left panels) Representative western blot analysis of the levels of SPDL1 protein upon knockdown of the indicated factors. (Right panels) Quantification of western blot analysis for three biological replicates. (F) Schematic representation of predicted base-pairing interactions for canonical U1 snRNA and for shifted U1 snRNAs with SPDL1 exon 3 5′ss and downstream intronic region. Bases in bold indicate sequences different from the canonical U1 sequence. (G) Quantification of SPDL1 exon 3 inclusion (PSI) upon transfection of wild type or variant U1 snRNAs, measured by RT-PCR and capillary electrophoresis in HeLa cells. Cells were cotransfected with a GFP plasmid to allow sorting of GPF-positive cells, which coexpress U1 snRNAs shown in F, 24 h after transfection. Data are represented as mean ± SD of three biological replicates and P-values were calculated by Student's t-test ([***] P < 0.001). (G) Clonogenic assay of GFP-positive HeLa cells sorted 24 h after cotransfection of GFP- and U1 snRNA-expressing plasmids shown in F. The number of colonies is normalized to that of control samples transfected only with the GFP plasmid (y-axis). Data are represented as mean ± SD of three biological replicates and P-values calculated by one-way ANOVA test ([***] P < 0.001).