Novel z-disc localized DUF4585 containing proteins and their contribution to sarcomere organization.

Introduction: Sarcomere Z-lines in cardiomyocytes function as hubs for transcript localization, translation, and protein interactions. Despite their critical role in sarcomere maintenance, many associated proteins remain unidentified. Using proximity labeling, we identified two proteins containing Domain of Unknown Function (DUF) 4585 in their C-terminus, localized to the Z-line.

Aims:
1. To investigate the elements responsible for the specific localization of DUF4585 proteins.
2. To explore the role of DUF4585 proteins in sarcomere organization and maintenance.

Methods: Expression of DUF4585-containing proteins in mouse tissues was quantified using qPCR. CRISPR/Cas9-based homology-independent targeted integration confirmed protein expression and Z-line localization. The impact of DUF4585 proteins on sarcomere structure was assessed by siRNA-mediated knockdown in cultured cardiomyocytes and gRNA-driven knockout in vivo. mRNA localization and translation were analyzed using smFISH and OPP assays. Localization elements were studied by expressing truncated protein fragments via Adeno virus.

Results: DUF4585 proteins were localized to the Z-line of sarcomeres. Knockdown in cardiomyocytes resulted in altered cell size and disrupted sarcomere organization, as shown by immunofluorescence. smFISH and OPP assays revealed changes in mRNA localization to the Z-line and disrupted translation. The C-terminal fragment of DUF4585 alone was sufficient for Z-line localization. In vivo, double knockout of DUF4585 caused severe cardiac myopathy, with disrupted sarcomere structure, altered costameric protein expression, and compensatory hypertrophy in non-transduced cells.

Conclusion: We identified novel DUF4585 proteins localized to the sarcomere and showed that the C-terminus regulates their Z-line localization. We showed that these proteins are essential for cardiomyocyte morphology, sarcomere structure, mRNA localization, and localized translation, both in vitro and in vivo, highlighting their importance in sarcomere maintenance.