1 Actinins are actin cross-linkers that are expressed in nearly all cells and harbor mutations in 2 heritable diseases. We deployed proximity-dependent biotinylation to identify actinin proximity 3 proteins and RNA transcripts in human cardiomyocytes to reveal new functions of the actin 4 cytoskeleton. We identified 285 proximity protein partners including unexpected effectors of 5 RNA-binding and metabolism, and interrogated dynamic partners using a sarcomere assembly 6 model. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein 7 associated with type 2 diabetes, interacted with the rod domain of actinin through its K 8 Homology domain. This interaction was necessary for actinin localization and translation of 9 electron transport chain transcripts, and oxidative phosphorylation. Diminished IGF2BP2 in 10 cardiac microtissues impaired contractile function in accord with diminished metabolic functions. 11 This actinin proximity protein and RNA study expands our functional knowledge of the actin 12 cytoskeleton, and uncovers new modes of metabolic regulation through interactions with RNA-13 binding proteins. 14 15 Main 16 Actinin proteins are ubiquitous spectrin family members that cross-link actin, and are important 17 for a myriad of cellular functions including cell adhesion, migration, contraction, and signaling 1 . 18 The four human actinin isoforms have distinct expression profiles (non-muscle ACTN1 and 19 ACTN4; cardiac muscle ACTN2; and skeletal muscle ACTN3), but share homologous amino 20 acid sequences that are organized into three structural domains-an actin-binding (AB) domain 21 composed of two calponin-homology domains, a central rod domain containing four spectrin-like 22 repeats (SR), and a calmodulin-like domain (CaM) containing two EF hand-like motifs 2 . While it 23is thought that actinin evolved initially to regulate the early eukaryotic actin-based cytoskeleton 3 , 24 it has acquired more elaborate functions in vertebrates, including a mechanical role in the 25 sarcomere, a specialized contractile system required for striated muscle function 3, 4 . Moreover, 21 microtissues with heart failure-associated mutations, twitch force was unaltered by Actinin-BirA* 22 expression (extended data Fig. 1b). We confirmed appropriate localization of Actinin-BirA* to the 23 Z-disk by both co-localization immunofluorescence (Fig.1d) and HA-immunoprecipitation assays 24 (Fig.1e). Actinin-BirA* interacted with the Z-disk protein titin-cap (TCAP), but not the sarcomere 25 M-line protein myomesin [22][23][24][25] , which supported appropriate localization. With the knowledge that 26 Confidential Manuscript 6 Actinin-BirA* functions similarly to unmodified actinin, we next optimized biotin supplementation