TBX3 Regulates Splicing <i>In Vivo</i>: A Novel Molecular Mechanism for Ulnar-Mammary Syndrome

<div><p>TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. <i>TBX3</i> mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins <i>in vivo</i> and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing <i>in vivo</i> and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that <i>TBX3</i> mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of <i>TBX3</i> mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.</p></div

Tbx3 regulates alternative splicing <i>in vitro</i>.

<p>A) Either pRHCglo (Control vector) or RHCGlo+T-box binding element (T-box vector) were co-transfected into HEK293 cells with Tbx3 expression vectors indicated above each lane. Splicing products were assayed by RT-PCR; positions of unspliced pre-mRNA and spliced products are indicated at left. Hprt control PCR products are shown below. B) Ability of Tbx3 mutant proteins to regulate T-box vector splicing. Symbols at bottom of panel summarize effect on splicing inhibition. b, baseline ratio of pre-mRNA to completely spliced mRNA; +, splicing inhibited over baseline; −, no inhibition. C) Immunoblot of control or TBX3 siRNA lysates probed for TBX3 and tubulin. D) Splicing products from the control minigene vector in the presence of Tbx3 or Tbx3 knockdown, assayed by RT-PCR. Schematics and positions of unspliced pre-mRNA (1746) and fully spliced (190) products are indicated at left. Baseline (b) ratio of unspliced to fully spliced mRNA from the control minigene vector is unchanged by overexpression of Tbx3 or knockdown of endogenous TBX3 in HEK293 cells. E) Splicing products from the control minigene vector in the presence of Tbx3 mutant proteins assayed by RT-PCR. In the absence of a TBE in the minigene, Tbx3 mutant proteins have no effect on the baseline ratio of unspliced to fully spliced mRNA.</p

TBX3 interacting proteins involved in mRNA splicing.

<p>* GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; UP: Uniprot.</p

TBX3 binds TBE-containing RNAs directly.

<p>A–E) EMSA assays of purified, MBP-conjugated Tbx3 with radiolabeled RNA probes whose sequences are listed at bottom of panels. C–E) RNA probes derived from <i>Nfkb1</i> intronic fragments (F1–F3). Black arrowheads: probe/protein complex; clear arrowheads: unbound probe. F–H) Shift and supershift RNA EMSA assays of endogenous TBX3; radiolabeled probe sequences are listed below panels. Probes were incubated +/− HEK293 lysate, +/− anti-TBX3 antibody, +/− TBX3 knockdown. F) RNA probe containing <u>GGUGU</u> motifs. Probe/protein complex (lane 5) supershifts with anti-TBX3 antibody (lanes 2, 3) and disappears with TBX3 knockdown (lane 6). TBX3+RNA complexes are indicated by blue arrowheads, anti-Tbx3 antibody supershifted TBX3+RNA complexes are indicated by red arrowheads and unbound probe by black arrowheads. Gray arrowhead indicates probe-protein complexes detected in the absence of TBX3 (lane 6). G) Mutation of the GGUGU to <u>CCUGU</u> results in formation of probe/protein complexes (lane 2) does not supershift with anti-TBX3 antibody (lane 3) and unaffected by TBX3 knockdown (lane 5). H) RNA probe containing <u>AGGUGUG</u> consensus TBE motif from <i>Nfkb1</i> Fragment 3. Probe/protein complex (lanes 2, 3, 5, blue arrowhead) supershifts with anti-TBX3 antibody (lanes 2, 3, red arrowheads) and decreases with TBX3 knockdown (lane 6). Gray arrowhead indicates probe-protein complexes detected in the absence of TBX3 (lane 6). Probe incubated with no lysate (F–H, lanes 1) or anti-TBX3 antibody alone (F–H, lanes 4) are additional negative controls. IgG negative control EMSAs are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004247#pgen.1004247.s005" target="_blank">Figure S5</a> G, H. I, J) Significantly over-represented sequence motifs identified by MEME on genomic regions flanking statistically significant AS events contain the 5′GGTG T-box core binding element.</p

Tbx3 binding partners associate with the <i>Nfkb1</i> mRNA that is alternatively spliced in response to Tbx3, and a subset require Tbx3 to bind this mRNA.

<p>A) RNA-IP with anti-Tbx3 on embryo lysates using antibodies against Tbx3 interacting proteins to test binding to the <i>Nfkb1</i> transcript which is differentially spliced in response to Tbx3 in mouse limb. Actin and Gapdh are negative controls. Note that not all Tbx3 interactors bind this mRNA. B) RIP/RT-PCR testing Tbx3 interactors for binding of the <i>Nfkb1</i> mRNA in <i>Tbx3</i> wild type (wt) and null (ko) murine embryonic fibroblasts (MEFs). Ddx3 requires Tbx3 to bind the <i>Nfkb1</i> mRNA (arrowhead) but hnrnpu does not. C) RT-PCR assay of splice variants in control, <i>Tbx3</i> and <i>Ddx3</i> siRNA knockdown MEFs. Knockdown of <i>Ddx3</i> results in the same alternative splicing of <i>Nfkb1</i> exon 11 as <i>Tbx3</i> knockdown, whereas knockdown of hnrnpu decreases the total amount of <i>Nfkb1</i> transcript but has no effect on exon 11 splicing. C′) <i>Gapdh</i> control RT-PCR.</p

Different Tbx3 protein domains are required for protein-protein interactions.

<p>Stable retroviral mediated knockdown of endogenous TBX3 in HEK293 cells followed by transfection of wild type and Tbx3 mutant proteins as diagrammed. Lysate IPs were assayed by IB with antibodies noted at bottom of panels. A) Immunoblot of control versus <i>TBX3</i> shRNA (KD) lysates. B) Schematic of Tbx3 mutant proteins. Confirmation of expression and successful IP of overexpressed Tbx3 proteins is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004247#pgen.1004247.s002" target="_blank">Figure S2</a>. C–E) Interactions of mutant Tbx3 proteins with DDX3, hnRNP C, and DDX17 tested by IP and western blotting. F–H) Tbx3 NLS is required for Tbx3 interactions with DDX3, DDX17 and hnRNP C. red arrowheads: interacting proteins; black arrowheads: IgG.</p

Tbx3 regulates alternative splicing <i>in vivo</i>.

<p>A, B, D, E) Screen shots from the Integrated Genome Viewer (IGV) comparing RNA-Seq reads obtained from wild type/control anterior (CA1, CA2) or posterior (or CP1, CP2) limb bud mRNA libraries with those from <i>Tbx3;Prx1Cre</i> mutants after conditional ablation of <i>Tbx3</i> in anterior or posterior limb bud mesenchyme (MA1, MA2 or MP1, MP2, respectively). Red arrowheads indicate positions of exons that are differentially spliced in a Tbx3-dependent manner. A) Loss of Tbx3 causes exclusion of <i>Dlg3</i> exons 8 and 9 in the mutant anterior compartment (MA1, MA2 red arrowheads). B) Loss of Tbx3 causes inclusion of <i>Dlg3</i> exons 8 and 9 in the mutant posterior compartment (MP1, MP2 red arrowheads). C) Schematic of splice variants and location of PCR primers used to detect different <i>Dlg3</i> isoforms by RT-PCR assay of control versus <i>Tbx3</i> mutant mRNAs (C′, Anterior and Posterior compartments, ctl, control; mt, mutant). D) Loss of Tbx3 causes exclusion of <i>Nfkb1</i> exon 11 in the mutant anterior compartment (MA1, MA2 red arrowheads). E) Loss of Tbx3 causes inclusion of <i>Nfkb1</i> exon 11 in the mutant posterior compartment (MP1, MP2 red arrowheads). F) Schematic of splice variants and location of PCR primers used to detect different <i>Nfkb1</i> isoforms by RT-PCR assay of control versus <i>Tbx3</i> mutant mRNAs (F′, Anterior and Posterior compartments, ctl, control; mt, mutant). G) Schematic of <i>Nfkb1</i> exon 11 minigenes in which exon 2 of pRHCglo was replaced by different <i>Nfkb1</i> genomic fragments (F1, F2, F3). H) Splicing products assayed by RT-PCR from the <i>Nfkb1</i> minigenes containing fragments F1, F2 or F3 in the presence of Tbx3 or after knockdown (KD). Only F3 confers differential splicing (lane 12). I) Schematic of splice variants obtained <i>in vivo</i> (sequence confirmed) in response to TBX3 knockdown. J) Splicing products assayed by RT-PCR from the <i>Nfkb1</i> F3 minigene in the presence of Tbx3 different Tbx3 mutant proteins. C-terminal mutants (lanes 3–6) result in alternate splicing similar to Tbx3 knockdown.</p

Metabolic Remodeling in Moderate Synchronous versus Dyssynchronous Pacing-Induced Heart Failure: Integrated Metabolomics and Proteomics Study

<div><p>Heart failure (HF) is accompanied by complex alterations in myocardial energy metabolism. Up to 40% of HF patients have dyssynchronous ventricular contraction, which is an independent indicator of mortality. We hypothesized that electromechanical dyssynchrony significantly affects metabolic remodeling in the course of HF. We used a canine model of tachypacing-induced HF. Animals were paced at 200 bpm for 6 weeks either in the right atrium (synchronous HF, SHF) or in the right ventricle (dyssynchronous HF, DHF). We collected biopsies from left ventricular apex and performed comprehensive metabolic pathway analysis using multi-platform metabolomics (GC/MS; MS/MS; HPLC) and LC-MS/MS label-free proteomics. We found important differences in metabolic remodeling between SHF and DHF. As compared to Control, ATP, phosphocreatine (PCr), creatine, and PCr/ATP (prognostic indicator of mortality in HF patients) were all significantly reduced in DHF, but not SHF. In addition, the myocardial levels of carnitine (mitochondrial fatty acid carrier) and fatty acids (12:0, 14:0) were significantly reduced in DHF, but not SHF. Carnitine parmitoyltransferase I, a key regulatory enzyme of fatty acid ß-oxidation, was significantly upregulated in SHF but was not different in DHF, as compared to Control. Both SHF and DHF exhibited a reduction, but to a different degree, in creatine and the intermediates of glycolysis and the TCA cycle. In contrast to this, the enzymes of creatine kinase shuttle were upregulated, and the enzymes of glycolysis and the TCA cycle were predominantly upregulated or unchanged in both SHF and DHF. These data suggest a systemic mismatch between substrate supply and demand in pacing-induced HF. The energy deficit observed in DHF, but not in SHF, may be associated with a critical decrease in fatty acid delivery to the ß-oxidation pipeline, primarily due to a reduction in myocardial carnitine content.</p></div

Metabolomic and proteomic profile of glucose metabolism.

<p>Data from Control, SHF, and DHF hearts. Detected metabolites and enzymes are indicated with bold font. Metabolome is presented in arbitrary units while proteome is presented as fold change compared to Control. Filled bars: metabolites. Open bars: proteins. G1-P: glucose 1-phosphate, G6-P: glucose 6-phosphate, F6-P: fructose 6-phosphate, F1,6-P: fructose 1,6-bisphosphate, GAP: glycealdehydo 3-phosphate, DHAP: dihydroxyacetone phosphate, 1,3-PG: 1,3-bisphosphoglycerate, 3-PG: 3-phosphoglycerate, 2-PG: 2-phosphoglycerate, PEP: phosphoenolpyruvate. P*<0.05.</p

Fatty acid catabolism.

<p>A schematic overview of fatty acid catabolism is presented with the levels of detected metabolites and relevant proteins in Control, SHF, and DHF. Detected metabolites and proteins are indicated with bold font. Blue color indicates enzymes involved in fatty acid oxidation. Metabolome is presented in arbitrary units while proteome is presented as fold change compared to Control. Filled bars: metabolites, Open bars: proteins. OCTN2: organic cation transporter novel type 2, FATP: fatty acid transport protein, FABP: fatty acid binding protein, CPT1: carnitine palmitoyltransferase I, CPT2: carnitine palmitoyltransferase II, CACT: carnitine O-acetyltransferase; DH: dehydrogenase; ETF: electron-transferring flavoprotein; ETFDH: electron transfer flavoprotein-ubiquinone oxidoreductase; CRAT: carnitine O-acetyltransferase. *P<0.05.</p