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 3

Rpsa knockdown disrupts localized translation in cultured cardiomyocytes.

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Rpsa knockdown disrupts localized translation in cultured cardiomyocyte...
(A) Gene expression analysis with RT-qPCR of Rpsa and Rpl38 following siRNA-mediated knockdown compared with control siRNA. Expression was normalized to Gapdh. n = 3, and 8 biological samples per experimental group pooled from 3 independent experiments. (B) Western blot analysis with densitometric analysis of RPSA protein expression showing an approximately 70% reduction in RPSA protein level compared with control following siRNA-mediated knockdown. n = 6 biological samples per experimental group from 3 independent experiments. (C) NRVMs were incubated with OPP to label nascent proteins 48 hours after treatment with negative control (top), Rpl38 (middle), or Rpsa (bottom) siRNAs. Representative images of staining for OPP-labeled nascent proteins (green), α-actinin (red), and DAPI (blue) show disruption of localized protein synthesis following Rpsa knockdown but not following Rpl38 knockdown or under control conditions. Scale bar: 10 μm. (D) Cartoon illustrating a structure with decreased homogeneity (top, score = 0.959) and a structure with increased homogeneity (bottom, score = 0.977). (E and F) Homogeneity scores for translation from the OPP signal (E) and for the sarcomeric structure from the α-actinin signal (F), showing increased homogeneity implying decreased localization following Rpsa knockdown but not following Rpl38 knockdown or control siRNAs. (G) Colocalization analysis between the OPP and the α-actinin signal showing significantly decreased colocalization between translation and the Z-line after knockdown of Rpsa. n = 79, 87, and 116 cells per group pooled from 4 independent experiments. **P ≤ 0.01, ****P ≤ 0.0001, by unpaired Student’s t test (A and B) and 1-way ANOVA test (E–G). Data are presented as individual values, with box plots displaying the median and 25th and 75th percentiles.