Desmoplakin and talin2 are novel mRNA targets of fragile X-related protein-1 in cardiac muscle

Abstract

Rationale: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied.

Objective: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish.

Methods and results: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis.

Conclusions: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Figure 1

Loss of FXR1 causes disruption of the inner dense plaque of desmosomes. In WT hearts (A & B), inner dense plaques are prominent, where intermediate filaments anchor to the membrane of the desmosome (double-headed red arrows). Loss of FXR1 in the KO hearts (C & D) results in reduced inner dense plaques at desmosomes (red arrowheads). Panels labeled A′–D′ are higher magnification views of the boxed areas in A–D. N=2 per genotype. Bars = 250 nm.

Figure 2

Loss of FXR1 leads to defects in sarcomere spacing and structure, and lateral adherence of myofibrils. Immunofluorescent localization of Z-disc titin shows even spacing and alignment of sarcomeres in WT hearts (A). Sarcomere spacing is erratic and shortened in Fxr1 KO hearts (1.31±0.40μm (N=240), compared to 1.68±0.18μm (N=150) in WT hearts (p < 0.05) (B, asterisks). Myofibrils also lose lateral bundling and “splay” out in the KO hearts (C, noted by #). Electron micrographs of WT hearts reveal evenly spaced Z-discs (arrowheads), organized thick filaments (arrows) within sarcomeres and clearly aligned myofibrils (D). In contrast, Z-discs (arrowheads in E & F) are variably broad, disorganized and unevenly spaced in KO hearts (E); disorganized thick filaments are apparent (F, arrows). Additionally, the lack of lateral bundling of myofibrils is observed in the KO hearts (F, noted by >). Bars in A–C = 15μm, D–F = 1μm.

Figure 3

Dsp and tln2 mRNAs are enriched in the FXR1 protein complex. (A) FXR1 was immunoprecipitated from adult mouse heart lysate (left lane) or the anti-FXR1 antibodies were competed with their antigenic peptide to establish specificity (right lane) (N=3). RNA isolated from immunoprecipitations was quantified and matched to amounts of total input RNA prior to qPCR analysis. Real-time PCR was used to assess the enrichment of candidate mRNA targets in the FXR1-IP compared to input RNA and control IP RNA (B). Beta-2-microglubulin (b2m) RNA is a negative control not associated with the FXR1 complex. Data shown as fold change (2^^{-(IPCt-InputCt)}) ± SD. * = reproducible >2-fold change. Fxr1, dsp and tln2 are enriched in the FXR1 complex 6.8-, 7.3-, and 13.1-fold, respectively.

Figure 4

Desmoplakin and talin2 protein levels are upregulated in Fxr1 KO mouse hearts. (A) Representative Western blots performed on WT and KO heart lysates for candidate targets enriched in the FXR1 protein complex: desmoplakin and talin2. (B) Quantification shows the percent increases in protein expression in the KO compared to the WT mouse hearts after normalization to GAPDH. Desmoplakin and talin2 protein levels are up in KO hearts 76±19% and 117±34%, respectively. (C) Total mRNA of target molecules dsp and tln2 are unchanged in KO compared to wild type hearts. (D) Representative Western blots of non-target molecules desmin and vimentin. (E) Non-target proteins desmin and vimentin are down regulated in KO hearts by 32±4% and 37±15%, respectively after normalization to GAPDH. Total mRNA of non-targets des and vim (F) are unchanged in KO compared to wild type hearts. Samples were normalized to Odc housekeeping gene and analyzed using the ΔΔCT method (2^^{-(ΔCTtarget-ΔCTodc)}). No notable alterations in total transcript levels of any candidate genes were detected, as defined by a reproducible >2-fold change. N=4 per genotype for both protein expression and total mRNA studies. Protein expression levels provided as mean ± SD. * = p < 0.05.

Figure 5

Talin2 protein localization expands beyond the typical costameric localization in Fxr1 KO mice. Sections of E18.5 hearts were stained for talin (green) and I-band titin (N2A, red). In WT hearts (A–C) talin is found in distinct puncta at costameres near the cell membrane (A, insert). Staining for talin in KO hearts in regions where the sarcomeric architecture looks similar to that of the WT (D–F) displays continuous staining along the length of the membrane in addition to occasional puncta in register with titin (white arrows). Talin localization in disrupted areas (G–I) exhibits staining in gaps lacking titin staining (asterisks). Bar = 15μm.

Figure 6

Localization of tln2 mRNA is not affected by the loss of FXR1. Sections of E18.5 hearts from WT and KO mice were probed for tln2 mRNA by Fluorescence In Situ Hybridization (FISH) (green). Hearts were co-stained for α-actinin (red). In WT (A–C), and KO (D–F) hearts tln2 mRNA localizes in discreet puncta amongst the myofibrils, relating closely with (arrows) and between (asterisks) Z-discs. Bar = 10μm.

Figure 7

FXR1 directly binds and represses the translation of dsp and tln2 mRNAs in vitro. (A) Data from dual reporter luciferase assays in COS-7 cells (N=6). Firefly luciferase containing Beta-2 microglobulin in the 3′UTR serves as control RNA that is not translationally repressed by FXR1. When the luciferase reporter contains the Fxr1 open reading frame (ORF), the dsp 3′UTR or the tln2 3′UTR, average luciferase activity is repressed to 31.6%, 53.1% and 56.7%, respectively (* = p < 0.01, mean ± SD). (B) Direct protein-RNA binding experiments show recombinant FXR1 binds biotin-dsp 3′UTR, and biotin-tln2 3′UTR RNA, but not biotin-Beta-2 microglubulin 3′UTR (as detected by probing Western Blots for FXR1, after incubation with immobilized biotin-RNA).