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Trbp regulates heart function through microRNA-mediated Sox6 repression

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Abstract

Cardiomyopathy is associated with altered expression of genes encoding contractile proteins. Here we show that Trbp (Tarbp2), an RNA-binding protein, is required for normal heart function. Cardiac-specific inactivation in mice of Trbp (TrbpcKO) caused progressive cardiomyopathy and lethal heart failure. Loss of Trbp function resulted in upregulation of Sox6, repression of genes encoding normal cardiac slow-twitch myofiber proteins and pathologically increased expression of genes encoding skeletal fast-twitch myofiber proteins. Remarkably, knockdown of Sox6 fully rescued the Trbp-mutant phenotype, whereas mice overexpressing Sox6 phenocopied TrbpcKO mice. Trbp inactivation was mechanistically linked to Sox6 upregulation through altered processing of miR-208a, which is a direct inhibitor of Sox6. Transgenic overexpression of Mir208a sufficiently repressed Sox6, restored the balance in gene expression for fast- and slow-twitch myofiber proteins, and rescued cardiac function in TrbpcKO mice. Together, our studies identify a new Trbp-mediated microRNA-processing mechanism in the regulation of a linear genetic cascade essential for normal heart function.

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Figure 1: Cardiac-specific Trbp knockout results in contraction defects in the heart.
Figure 2: Genome-wide identification of dysregulated mRNAs in TrbpcKO hearts.
Figure 3: Reintroduction of Trbp in TrbpcKO hearts rescues cardiac defects.
Figure 4: The function of Trbp is mediated by Sox6 in the heart.
Figure 5: Genome-wide identification of dysregulated miRNA species in TrbpcKO hearts.
Figure 6: MiR-208a is a Trbp target and mediates its function in TrbpcKO hearts.

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Acknowledgements

We thank D. Clapham and members of the Wang laboratory for advice and support. We thank R. Espinoza-Lewis, G. Wang and F. Gu for technical support. We thank F.F. Wang for careful reading of the manuscript. Work in the Wang laboratory is supported by the March of Dimes Foundation, the Muscular Dystrophy Association and the US National Institutes of Health (HL085635 and HL116919). M.K. was supported by Banyu Life Science Foundation International.

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Contributions

J.D. and D.-Z.W. conceived the project, designed the experiments, analyzed the data and wrote the manuscript. J.D. generated and characterized the Trbp-mutant mice and performed molecular biology experiments. J.D., L.M. and P.Z. contributed to targeting vector construction and Southern blotting. J.D. and J.C. contributed to echocardiographic data acquisition and analysis. Y.W. analyzed the RNA-seq data. J.D., X.H., M.N. and Z.-L.D. contributed to morphological and histological data acquisition and analysis. J.D., M.K. and Z.L. contributed to AAV preparation and administration. W.T.P. supervised AAV preparation and administration and reviewed the manuscript.

Corresponding author

Correspondence to Da-Zhi Wang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Generation of Trbp knockout mice.

(a) Schematic of the Trbp knockout strategy. (b) Southern blot and PCR assays to confirm the desired recombination events. (c) qRT-PCR of Trbp expression in the indicated organs of Trbp-null (Trbp−/−) and wild-type (WT) mice. n = 3; **P < 0.01. (d) Left, Kaplan-Meier survival curves of Trbp−/− and WT mice. Middle, gross morphology of 2-week-old Trbp−/− and WT mice. Right, body weight of 2-week-old Trbp−/− and WT (+/+) mice. n = 6; **P < 0.01.

Supplementary Figure 2 Expression of cell cycle markers in the heart.

qRT-PCR documenting the expression of the indicated cell cycle marker genes in the hearts of TrbpcKO and control mice at various stages. P2.5, postnatal day 2.5; 2wk, 2 weeks after birth; 1mo, 1 month after birth. n = 3; NS, not significant; *P < 0.05, **P < 0.01.

Supplementary Figure 3 Overexpression of Hopx fails to rescue the defects of TrbpcKO mice.

(a) Expression of Hopx in the hearts of 2-week-old TrbpcKO and control mice assayed with qRT-PCR. n = 3; **P < 0.01. (b) Western blot to detect Hopx and Trbp proteins in the hearts of 2-week-old mice. Gapdh served as a loading control. (c) Kaplan-Meier survival curves of control/AAV-Luc, TrbpcKO/AAV-Luc, Control/AAV-Hopx and TrbpcKO/AAV-Hopx (TrbpcKO/AAV-Luc versus TrbpcKO/AAV-Hopx, P > 0.05). (d) Left-ventricle internal dimension at systolic (LVID;s) and fractional shortening (FS%) of TrbpcKO and control mice after AAV-Hopx or AAV-Luc injection. n = 6–17; NS, not significant; **P < 0.01.

Supplementary Figure 4 Sox6 expression is regulated by Trbp in the heart.

(a) Expression of Sox6 in the hearts of TrbpcKO and control mice at the indicated time points. n = 3; *P < 0.05, **P < 0.01. (b) Expression of Sox6 in the hearts of 1-month-old TrbpcKO and control mice injected with AAV-Trbp or AAV-Luc control. n = 3; *P < 0.05, **P < 0.01. (c) Gross morphology and histology of heart samples from 1-month-old wild-type mice injected with AAV-Sox6 or AAV-Luc control. Scale bars, 1.0 mm.

Supplementary Figure 5 Expression of miR-208a and miR-499 in the heart.

(a) qRT-PCR of pre-miR-208a in the hearts of TrbpcKO and control mice at the indicated time points. n = 3; **P < 0.01. (b) Quantification of the expression of pre-miR-208a in the hearts of 1-month-old TrbpcKO and control mice injected with AAV-Trbp or AAV-Luc control. (c) Quantification of the expression of pre-miR-208a in the hearts of 1-month-old TrbpcKO and control mice injected with AAV-Sox6 or AAV-Luc control. (d) Quantification of the expression of pre-miR-208a in the hearts of 1-month-old TrbpcKO, control, miR-208aTG and TrbpcKO; miR-208aTG mice. (e) qRT-PCR of miR-499 in the hearts of 1-month-old TrbpcKO and control mice injected with AAV-Trbp or AAV-Luc control. n = 3; **P < 0.01.

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Ding, J., Chen, J., Wang, Y. et al. Trbp regulates heart function through microRNA-mediated Sox6 repression. Nat Genet 47, 776–783 (2015). https://doi.org/10.1038/ng.3324

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