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Identifying Cardiac Actinin Interactomes Reveals Sarcomere Crosstalk with RNA-Binding Proteins

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Identifying Cardiac Actinin Interactomes Reveals Sarcomere Crosstalk with RNA-Binding Proteins bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title: Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA- 2 binding proteins. 3 4 Authors: 5 Feria A. Ladha M.S.1, Ketan Thakar Ph.D.2*, Anthony M. Pettinato B.S.1*, Nicholas 6 Legere B.S.2, Rachel Cohn M.S.2, Robert Romano B.S.1, Emily Meredith B.S.1, Yu-Sheng 2 1,2,3 7 Chen Ph.D. , and J. Travis Hinson M.D. 8 9 Affiliations: 10 1University of Connecticut Health Center, Farmington, CT 06030, USA 11 2The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA 12 3Cardiology Center, UConn Health, Farmington, CT 06030, USA 13 *These authors contributed equally 14 15 Lead Contact: Correspondence: 16 J. Travis Hinson, MD 17 UConn Health and The Jackson Laboratory for Genomic Medicine 18 10 Discovery Drive, Farmington, CT 06032 19 860-837-2048 (t) | 860-837-2398 (f) | [email protected] | [email protected] 20 21 Word count: 7,641 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Abstract 2 Rationale: Actinins are actin cross-linkers that are ubiquitously expressed and harbor 3 mutations in heritable diseases. The predominant cardiac actinin is encoded by ACTN2, 4 which is a core structural component of the sarcomere Z-disk. ACTN2 is required for 5 sarcomere function through complex interactions with proteins involved in sarcomere 6 assembly, cell signaling and transcriptional regulation. However, there remain critical 7 gaps in our knowledge of the complete and dynamic cardiac actinin interactome, which 8 could reveal new insights into sarcomere biology. 9 Objective: We sought to examine the cardiac actinin interactome through sarcomere 10 assembly in human cardiomyocytes. 11 Methods and Results: We utilized CRISPR/Cas9, induced pluripotent stem cell 12 technology and BioID to reveal cardiac actinin protein interactions in human 13 cardiomyocytes. We identified 324 cardiac actinin proximity partners, analyzed networks, 14 and studied interactome changes associated with sarcomere assembly. We focused 15 additional studies on unexpected actinin interactions with effectors with RNA-binding 16 functions. Using RNA immunoprecipitation followed by sequencing, we determined that 17 RNA-binding partners uncovered by actinin BioID were bound to gene transcripts with 18 electron transport chain and mitochondrial biogenesis functions. Mammalian two-hybrid 19 studies established that IGF2BP2, an RNA-binding protein associated with type 2 20 diabetes, directly interacted with the rod domain of actinin through its K Homology 21 domain. IGF2BP2 was necessary for electron transport chain transcript localization to 22 vicinal RNA-binding proteins, mitochondrial mass and oxidative metabolism. IGF2BP2 23 knockdown also impaired sarcomere function in a cardiac microtissue assay. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Conclusions: This study expands our functional knowledge of cardiac actinin, uncovers 2 new sarcomere interaction partners including those regulated by sarcomere assembly, 3 and reveals sarcomere crosstalk with RNA-binding proteins including IGF2BP2 that are 4 important for metabolic functions. 5 6 Introduction 7 Actinin proteins are ubiquitous spectrin family members that cross-link actin, and are 8 important for myriad cell functions including adhesion, migration, contraction, and 9 signaling1. The four human actinin isoforms have distinct expression profiles (non-muscle 10 ACTN1 and ACTN4; cardiac muscle ACTN2; and skeletal muscle ACTN3), but share 11 homologous amino acid sequences that are organized into three structural domains—an 12 actin-binding (AB) domain composed of two calponin-homology domains, a central rod 13 domain containing four spectrin-like repeats (SR), and a calmodulin-like domain (CaM) 14 containing two EF hand-like motifs2. While it is thought that actinin evolved initially to 15 regulate the early eukaryotic actin-based cytoskeleton3, it has acquired more elaborate 16 functions in vertebrates, including a mechanical role in the sarcomere, a specialized 17 contractile system required for striated muscle function3, 4. Moreover, normal actinin 18 function is important for multiple human organ systems as inheritance of ACTN1, ACTN2, 19 and ACTN4 mutations have also been linked to congenital macrothrombocytopenia, 20 hypertrophic cardiomyopathy, and focal segmental glomerulosclerosis of the kidney, 21 respectively5-7. 22 In the cardiac sarcomere, alpha actinin-2 (referred henceforth as actinin) is a major 23 structural component of the Z-disk, where it is essential for sarcomere assembly and 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 function8. Actinin regulates sarcomere assembly by providing a scaffold for protein- 2 protein interactions (PPIs) such as with titin (TTN) and actin through CaM and AB 3 domains, respectively8-10. Secondary to these interactions, the thin and thick filament 4 sarcomere structures are organized to promote twitch contraction. It has also been 5 observed that while the Z-disk is one of the stiffest sarcomere structures, actinin 6 demonstrates remarkably dynamic and complex molecular interactions such as with the 7 actin cytoskeleton that are necessary for normal myocyte function11. Moreover, actinin 8 interaction partners have also been described to exit the sarcomere to execute critical 9 roles in cell signaling, protein homeostasis and transcriptional regulation12. Secondary to 10 the vast repertoire and dynamics of actinin molecular interactions, there remain extensive 11 gaps in our knowledge of not only how actinin regulates sarcomere assembly and 12 function, but also within other cellular contexts enriched for actinin expression such as at 13 focal adhesions and cell surface receptors in both striated and non-striated cells that may 14 inform how actinin mutations cause human disease13, 14. 15 Here, we examine the actinin interactome in CRISPR/Cas9-engineered human 16 cardiomyocytes using proximity-dependent biotinylation (BioID). This study employs 17 cardiomyocytes (iPSC-CMs) differentiated from induced pluripotent stem cells (iPSCs) 18 that express actinin fused to the promiscuous biotinylating enzyme BirA* from the ACTN2 19 locus to provide in vivo actinin expression levels and localization15. We identify a list of 20 actinin neighborhood proteins including those that are dynamically regulated by 21 sarcomere assembly to reveal molecular insights into this process. Our study uncovers 22 unexpected actinin interactions with RNA-binding proteins including IGF2BP2 and 23 specific RNA transcripts bound to these vicinal RNA-binding proteins including electron 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 transport chain and mitochondrial biogenesis factors. We identify how this process is 2 essential for electron transport chain and sarcomere contractile functions. Our study also 3 provides a list of molecules that can be studied to reveal additional insights into sarcomere 4 dynamics and unexpected functions of actinin. 5 6 Methods 7 Detailed methods are provided in the Supplemental Materials. Statistical tests and 8 samples sizes are described in each figure legend. The data that support the findings of 9 this study are available from the corresponding author upon reasonable request. 10 iPSC-CM Differentiation and CRISP/Cas9 studies 11 All stem cell experiments were performed using PGP1 iPSCs (Coriell #GM23338). iPSC- 12 CMs were directly differentiated by sequential modulation of Wnt/β-catenin signaling, as 13 previously described16, 17. For BioID studies, control and Actinin-BirA* iPSC-CMs were 14 maintained in DMEM (Gibco 11965092) supplemented with homemade biotin-free B27 15 following metabolic enrichment18, 19. For proximity-labeling experiments, control and 16 Actinin-BirA* iPSC-CMs were treated with 50 μM biotin for 24 hours followed by a 2-hour 17 washout phase in biotin-free media before collection. 18 Genome editing was performed utilizing a CRISPR/Cas9 protocol adapted from 19 previous studies17, 20. iPSCs were electroporated with pCas9-GFP (Addgene 44719), 20 appropriate hU6-driven sgRNA, and HR targeting vector to generate ACTN2-BirA* knock- 21 in iPSCs. Isogenic knockout of TNNT2 was performed similarly, but in the absence of an 22 HR vector and with GFP-based FACS enrichment in place of antibiotic selection. 23 Streptavidin immunoprecipitation and Quantitative proteomics 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted May 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
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