Relative mRNA expression of alternative <i>Ank3</i> first exons.

<p>(<b>A</b>) Diagram of alternative splicing of 5 first exons of the <i>Ank3</i> gene. Gray dashed lines represent rare alternative spliced junctions (<1%). (<b>B</b>) qt-PCR analysis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression (measured as cycle threshold number) in ventricle, atria, skeletal muscle, brain, and kidney from three mice (labeled M1–M3). (<b>C</b>) Relative mRNA expression of alternative first exons was measured in heart (ventricle and atria), skeletal muscle, brain, and kidney by qt-PCR analysis. Bar graphs represent technical replicates of qt-PCR samples and error bars represent standard deviations. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparison test (*** <i>p</i>-value ≤0.001, ns: not significant) to assess the significance of expression differences between different splice variants (i.e. 1a vs. 1b).</p

Exon Organization and Novel Alternative Splicing of <i>Ank3</i> in Mouse Heart

<div><p>Ankyrin-G is an adaptor protein that links membrane proteins to the underlying cytoskeletal network. Alternative splicing of the <i>Ank3</i> gene gives rise to multiple ankyrin-G isoforms in numerous tissues. To date, only one ankyrin-G isoform has been characterized in heart and transcriptional regulation of the <i>Ank3</i> gene is completely unknown. In this study, we describe the first comprehensive analysis of <i>Ank3</i> expression in heart. Using a PCR-based screen of cardiac mRNA transcripts, we identify two new exons and 28 alternative splice variants of the <i>Ank3</i> gene. We measure the relative expression of each splice variant using quantitative real-time PCR and exon-exon boundary spanning primers that specifically amplify individual <i>Ank3</i> variants. Six variants are rarely expressed (<1%), while the remaining variants display similar expression patterns in three hearts. Of the five first exons in the <i>Ank3</i> gene, exon 1d is only expressed in heart and skeletal muscle as it was not detected in brain, kidney, cerebellum, and lung. Immunoblot analysis reveals multiple ankyrin-G isoforms in heart, and two ankyrin-G subpopulations are detected in adult cardiomyocytes by immunofluorescence. One population co-localizes with the voltage-gated sodium channel Na_V1.5 at the intercalated disc, while the other population expresses at the Z-line. Two of the rare splice variants excise a portion of the ZU5 motif, which encodes the minimal spectrin-binding domain, and these variants lack β-spectrin binding. Together, these data demonstrate that <i>Ank3</i> is subject to complex splicing regulation resulting in a diverse population of ankyrin-G isoforms in heart.</p></div

Relative mRNA expression of alternative <i>Ank3</i> transcripts encoding ankyrin-G MBD.

<p>(<b>A</b>) Diagram of <i>Ank3</i> alternative splicing in ankyrin-G MBD. (<b>B</b>) Expression of <i>Ank3</i> mRNA transcripts ± exon 16 or ± exon 18 was measured in three mouse hearts using qt-PCR analysis with exon-exon boundary spanning primers. Bar graphs represent technical replicates of qt-PCR samples and error bars represent standard deviations. Statistical analysis was performed with unpaired Student’s T-test (*** <i>p</i>-value ≤0.001).</p

Exon organization and alternative splicing of <i>Ank3</i> gene.

<p>(<b>A</b>) Diagram of ankyrin-G exon organization and nine overlapping PCR primer sets used to amplify cardiac-specific <i>Ank3</i> alternative transcripts. Ankyrin functional domains are indicated. MBD: membrane-binding domain, SBD: spectrin-binding domain, DD: death domain, CTD: C-terminal domain. The large (7694 bp) brain-specific exon corresponds to exon 38. (<b>B</b>) Diagram of alternative <i>Ank3</i> splice variants encoding the MBD, SBD, and CTD. In the MBD, 5 alternative first exons (1a-1e) were detected. Lengths of intervening intronic sequences are labeled in kilobases (k). In the SBD, two novel exons (27 and 30) were identified and exon numbering has been adjusted accordingly (exon 40 is now the brain-specific exon). Exon 25 (in red) encodes the alternative start site for truncated ankyrin-G isoforms that lack MBDs (e.g. AnkG-107). In the CTD, alternative splicing of exons 46–49 (highlighted in gray) was not addressed in this study. (<b>C</b>) Full-length exon-exon boundary spanning primers are necessary to amplify PCR products: <i>Ank3</i> splice junctions of exons 26/28 and 26/t28 (truncated) (first panel) and exons 43/44 and 43/t44 (second panel).</p

<i>Ank3</i> exon organization.

<p>Table includes current and previous exon numbers (NM_146005.3), adjacent nucleotide sequences to 5’- and 3’- splice junctions, exon length (and length of coding sequence CDS for first exons), intron length, functional domain encoded by the exon, and any additional comments. NCBI accession numbers for the first exons are as follows: exon 1a: BB614215, exon 1b: BX520427, exon 1c: CD802881, exon 1d: DV047480, exon 1e: AI614790, and exon 25: BB628284.</p><p><i>Ank3</i> exon organization.</p

<i>Ank3</i> primer sets.

<p><i>Ank3</i> primer sets.</p

Alternative ankyrin-G SBD isoforms do not bind β-spectrin.

<p>(<b>A</b>) Diagram of <i>Ank3</i> exon organizations for two rare ankyrin-G SBD variants (ΔE31 and ΔE28–31). Both variants lack a portion of the ZU5 motif. (<b>B</b>) β1-spectrin and β2-spectrin (repeats 13 to 17) binding to ankyrin-G SBDs (wildtype, Δ31, Δ28–31) was measured by <i>in vitro</i> binding assays. Label is the unbound <i>in vitro</i> translated SBD products. (<b>C</b>) Coomassie Blue Stain demonstrates equal loading of GST-fusion proteins β1- and β2-spectrins.</p

Subcellular localization of ankyrin-G CTD isoforms in neonatal cardiomyocytes.

<p>(<b>A</b>) Diagram of lenti-viral GFP-tagged CTD variants with full-length or truncated exon 44. (<b>B</b>) GFP immunoblot analysis demonstrating expression of lenti-viral constructs. (<b>C</b>) Immunofluorescent co-localization of GFP-tagged CTD variants (fl-E44-GFP and tr-E44-GFP) with Z-line resident protein α-actinin in neonatal cardiomyocytes. Bottom panels represent localization of endogenous ankyrin-G in reference to α-actinin. Scale bar represents 10 microns.</p