Localized translation and sarcomere maintenance requires ribosomal protein SA in mice
Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat
Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat
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Research Article Cardiology

Localized translation and sarcomere maintenance requires ribosomal protein SA in mice

Abstract

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified ribosomal protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9-expressing mice, along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mislocalization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrated that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

Authors

Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat

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Figure 7

Rpsa knockout disrupts sarcomeric ribosome localization in vivo.

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Rpsa knockout disrupts sarcomeric ribosome localization in vivo. (A and...
(A and B) Dual smFISH with probes for ribosomal 18S RNA (white) and Cre mRNA (red) was used to visualize ribosomes and targeted cardiomyocytes, respectively. Representative images show sarcomeric cross-striated pattern of ribosomes localization in both targeted and untargeted cardiomyocytes in negative control mice (A). In Rpsa-knockout mice, the sarcomeric localization of ribosomes is lost in targeted Cre-positive cardiomyocytes, while untargeted cells are normal (B). High-magnification images (magnified 6.5 times the original image) of the boxed areas showing untargeted cytoplasmic Cre mRNA–negative cell (gray rectangle) and targeted cytoplasmic Cre mRNA–positive cell (light red rectangle) for both the negative control (A) and Rpsa-knockout (B) hearts. Scale bar: 10 μm. (C and D) The intensity of ribosomal 18S RNA FISH signal along the cardiomyocytes in the boxed areas shows sarcomeric periodicity in both the targeted (light red line) and untargeted (gray line) cardiomyocytes in negative control mice (C). In Rpsa-knockout mice (D), the sarcomeric periodicity of ribosomes is lost in the targeted Cre-positive cardiomyocyte (light red line), while the untargeted cell has normal periodicity (gray line). (E) Line-scan quantification confirms the loss of localized sarcomeric 18S signal in Rpsa-knockout cardiomyocytes. n = 2 mice per experimental group; n = 49 and 83 cells. ****P ≤ 0.0001, by Student’s t test. Data are presented as individual values, with box plot displaying the median with 25th and 75th percentiles.